High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3022-3022
Author(s):  
Jonathan Scott Rink ◽  
Sol Misener ◽  
Osman Cen ◽  
Shuo Yang ◽  
Leo I. Gordon ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Density Lipoprotein ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Cholesterol Homeostasis ◽  
B Cell Lymphomas ◽  
Bcr Signaling

Abstract Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas. Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk. Results: The Burkitt's lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death. Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. Disclosures Thaxton: Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

Scavenger Receptor Type B1 Is Essential for High Density Lipoprotein Nanoparticle Induced B-Cell Lymphoma Cell Death

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2756-2756 ◽  
Author(s):  
Jonathan Scott Rink ◽  
Shuo Yang ◽  
Osman Cen ◽  
Fei Ying ◽  
Young Kwang Chae ◽  
...  
Keyword(s):  
Cell Death ◽  
High Density Lipoprotein ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Density Lipoprotein ◽  
B Cell Lymphoma ◽  
High Density ◽  
Lymphoma Cell ◽  
B Cell Lymphomas

Abstract Introduction: We recently reported that biomimetic, synthetic high-density lipoprotein nanoparticles (HDL NP), similar to natural HDL in size, shape, charge, and composition potently induce apoptosis in human B cell lymphoma cell lines in vitro, and in vivo, without adversely affecting primary hepatocytes or macrophages (Yang et al. 2013. PNAS 110(7): 2511-6). We showed that HDL NPs bind to the high-affinity HDL receptor, scavenger receptor type B-1 (SR-B1), expressed by lymphoma cells, and may play a functional role in apoptosis of B cell lymphomas. Methods: We analyzed tissue microarrays (TMAs) from patients with non-Hodgkin's lymphoma (NHL) for SR-B1 expression. In silico analyses of gene microarray datasets from the publicly available database Oncomine were conducted to investigate SR-B1 expression in lymphoma at the mRNA level. The B cell lymphoma cell lines Ramos (Burkitt's lymphoma), SUDHL4 (diffuse large B cell lymphoma) and HF-1 (transformed follicular lymphoma) were used to investigate the requirement for SR-B1 in HDL NP induced cell death. A blocking antibody, and stably expressed shRNA targeting SR-B1 expression were used to inhibit HDL NP interactions with SR-B1. Results: SR-B1 was overexpressed in a subpopulation of NHL represented in available TMAs. At the mRNA level, SR-B1 was up-regulated in 20% of lymphoma data sets (6 of 30). These data further confirm SR-B1 as a potential target of therapeutic intervention in B cell lymphomas. Antibody blockade of SR-B1 in the SR-B1+ cell lines Ramos, SUDHL4, and HF-1 prevented HDL NP induced cell death in a dose dependent manner (Figure 1A). Stable knockdown of SR-B1 by shRNA in Ramos cells also protected against HDL NP induced cell death compared with wild type and scrambled shRNA controls (Figure 1B). HDL NP induced cholesterol efflux in Ramos, SUDHL4, and HF-1 cells was reduced by the SR-B1 blocking antibody, or SR-B1 knockdown, further supporting that the HDL NP binds SR-B1. Conclusion: SR-B1 is expressed in primary B cell lymphomas, and interference of HDL NP interaction with SR-B1, through antibody blockade or knockdown of SR-B1, abrogated HDL NP induced cell death in multiple B cell lymphoma cell lines. Taken together, our data demonstrate the requirement of SR-B1 in HDL NP induced lymphoma cell death, and provide a rationale to pursue HDL NPs as potent therapy for B cell lymphomas in cases that express SR-B1. Figure 1. Antibody blockade (A) and shRNA knockdown (B) of SR-B1 prevents HDL NP induce cell death. Figure 1. Antibody blockade (A) and shRNA knockdown (B) of SR-B1 prevents HDL NP induce cell death. Disclosures Gordon: Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending; Northwestern University: Employment. Thaxton:Aurasense: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: High Density Lipoprotein Nanoparticles for Lymphoma.

Notch-Regulated Enhancers in B-Cell Lymphoma Activate MYC and Potentiate B-Cell Receptor Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 457-457
Author(s):  
Russell J.H. Ryan ◽  
Jelena Petrovic ◽  
Dylan Rausch ◽  
Caleb Lareau ◽  
Winston Lee ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Rna Seq ◽  
Bcr Signaling ◽  
Notch Activation

Abstract Gain-of-function mutations in Notch receptor genes occur in 10-15% of cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and are associated with inferior clinical outcomes. Nearly all Notch mutations reported in B cell tumors lead to loss of the C-terminal negative regulatory PEST domain and result in stabilization of the activated form of Notch (intracellular Notch [ICN]), whereas mutations that lead to ligand-independent Notch activation (which are common in T cell acute lymphoblastic leukemia [T-ALL]) are rare. ICN can be detected in tumor cells within lymph nodes of >80% of patients with CLL, suggesting that Notch may have a broader oncogenic role than the incidence of Notch mutations would suggest. However, the downstream targets of Notch in B-cell tumors have not been identified. We used a gamma-secretase inhibitor (GSI) washout strategy to determine the immediate, direct effects of Notch activation in three MCL cell lines with Notch gain-of-function mutations, including two cell lines with unusual Notch gene rearrangements that lead to ligand-independent Notch activation, as well as a third line with a Notch PEST domain mutation in which signaling was activated with recombinant Notch ligand. Using these models, we identified likely direct target genes and their associated genomic Notch response elements using RNA-seq and ChIP-Seq in the Notch-on and Notch-off states. Most of these response elements corresponded to long-range enhancers that showed Notch-dependent changes in H3K27 acetylation, and were bound by components of the Notch transcription complex (NTC) in both cell lines. We confirmed these associations by performing ChIP-Seq on primary CLL and MCL biopsies, and by identifying specific looping interactions with Notch target gene promoters in public genome-wide proximity ligation datasets (RNA Pol2 ChIA-PET) from a lymphoblastoid cell line expressing the EBV-encoded Notch surrogate protein EBNA2. MYC was among the most strongly Notch-activated genes in Notch-dependent MCL cell lines and was associated with NTC binding at two B cell-specific 5' enhancers distinct from the Notch-dependent MYC enhancer previously identified in T-ALL. MCL cell line proliferation was blocked by Cas9 nuclease or epigenetic repressors targeting the 5' MYC enhancers, whereas cells were rescued from Notch inhibition by GSI via transduction with MYC. Gene set enrichment analysis of other direct Notch target genes identified in MCL models showed enrichment for regulators of B cell receptor (BCR) signaling, including the Src family kinase genes FYN, LYN, and BLK, and the signaling complex adaptor BLNK, as well as regulators of CD40 and cytokine signaling. RNA-seq analysis of primary CLL lymph node biopsies revealed significantly higher expression of many Notch target genes in biopsies with high levels of ICN. To functionally validate Notch target genes in primary tumors, we co-cultured CLL and MCL cells obtained from peripheral blood with Notch ligand-expressing stromal cells in the presence ("notch off") or absence ("notch on") of GSI, and demonstrated increased expression of Notch target genes, including MYC, in the "notch-on" cells. Furthermore, "notch-on" CLL cells showed increased phosphorylation of the BCR signaling intermediates SYK and PLCg2 upon BCR crosslinking compared to GSI-treated cells. Finally, we validated Notch-dependent regulation of target genes in vivo in a patient-derived xenograft model of NOTCH1-mutant MCL. Notch target gene expression was significantly higher in MCL cells within the spleen versus bone marrow or blood, but was markedly reduced in animals treated for five days with GSI. Additional xenograft studies are ongoing, and will be described at the meeting. Our data link active Notch signaling to two well-characterized oncogenic drivers in B cell lymphoma, MYC and BCR signaling, and may have important implications for the development of treatment strategies involving Notch antagonists and other targeted therapeutics, such as BCR targeting agents. Disclosures Weinstock: Novartis: Consultancy, Research Funding.

scholarly journals SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2230-2237 ◽  
Author(s):  
Linfeng Chen ◽  
Stefano Monti ◽  
Przemyslaw Juszczynski ◽  
John Daley ◽  
Wen Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Primary Tumors ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Survival Signals

The role of B-cell receptor (BCR)–mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igα/β immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as “BCR-type” tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

scholarly journals The B-Cell Receptor Is Required for Optimal Viability, Growth, and Chemotherapy Resistance of Diffuse Large B-Cell Lymphoma Cell Lines of the Germinal Center B-Cell Subtype

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 493-493 ◽  
Author(s):  
Ondrej Havranek ◽  
Stefan Koehrer ◽  
Justin M Comer ◽  
Zhiqiang Wang ◽  
Jingda Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Chemotherapy Resistance ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bcr Signaling ◽  
Relative Decline

Abstract Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL. Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting. Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells. GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO. Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways. Table A. B. Figure 1. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1B Figure 1B. Figure 2 Cell cycle changes with BCR KO in OCI-Ly7. Figure 2. Cell cycle changes with BCR KO in OCI-Ly7. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ibrutinib in B-cell lymphoma: single fighter might be enough?

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Chao Xue ◽  
Xin Wang ◽  
Lingyan Zhang ◽  
Qingyuan Qu ◽  
Qian Zhang ◽  
...  
Keyword(s):  
B Cell ◽  
Signaling Pathway ◽  
Cell Lymphoma ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
High Response Rate ◽  
Main Body ◽  
Combination Strategies ◽  
Bcr Signaling

Abstract Background In recent years, the B cell receptor (BCR) signaling pathway has become a “hot point” because it plays a critical role in B-cell proliferation and function. Bruton’s tyrosine kinase (BTK) is overexpressed in many subtypes of B-cell lymphoma as a downstream kinase in the BCR signaling pathway. Ibrutinib, the first generation of BTK inhibitor, has shown excellent antitumor activity in both indolent and aggressive B-cell lymphoma. Main body Ibrutinib monotherapy has been confirmed to be effective with a high response rate (RR) and well-tolerated in many B-cell lymphoma subgroups. To achieve much deeper and faster remission, combination strategies contained ibrutinib were conducted to evaluate their synergistic anti-tumor effect. Conclusions For patients with indolent B-cell lymphoma, most of them respond well with ibrutinib monotherapy. Combination strategies contained ibrutinib might be a better choice to achieve deeper and faster remission in the treatment of aggressive subtypes of B-cell lymphoma. Further investigations on the long-term efficacy and safety of the ibrutinib will provide novel strategies for individualized treatment of B-cell lymphoma.

Targeting B-cell receptor and PI3K signaling in diffuse large B-cell lymphoma

Blood ◽  
2021 ◽  
Author(s):  
Wendan Xu ◽  
Philipp Berning ◽  
Georg Lenz
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Genetic Alterations ◽  
B Cell Receptor ◽  
Special Focus ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category comprising distinct molecular subtypes characterized by diverse genetic aberrations that dictate patient outcome. As roughly one-third of DLBCL patients are not cured by current standard chemo-immunotherapy a better understanding of the molecular pathogenesis is warranted to improve outcome. B-cell receptor (BCR) signaling is crucial for the development, growth and survival of both normal and a substantial fraction of malignant B-cells. Various analyses revealed genetic alterations of central components of the BCR or its downstream signaling effectors in some subtypes of DLBCL. Thus, BCR signaling and the downstream NF-κB and PI3K cascades have been proposed as potential targets for the treatment of DLBCL patients. As one of the main effectors of BCR activation, PI3K mediated signals play a crucial role in the pathogenesis and survival of DLBCL. In this review, we summarize our current understanding of BCR signaling with a special focus on the PI3K pathway in DLBCL and how to utilize this knowledge therapeutically.

scholarly journals Establishment of two Epstein-Barr virus negative Burkitt cell lines from a patient with AIDS and B-cell lymphoma

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1255-1260 ◽  
Author(s):  
A Ganser ◽  
C Carlo-Stella ◽  
CR Bartram ◽  
T Boehm ◽  
G Heil ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Immunoglobulin Gene ◽  
Barr Virus ◽  
Epstein Barr ◽  
Immunodeficiency Syndrome

Abstract To analyze the pathogenesis of B-cell lymphomas in patients with acquired immunodeficiency syndrome (AIDS), we studied two cell lines, Es I and Es III, established from one such lymphoma for the presence of sequences of the Epstein-Barr virus (EBV) and the human immunodeficiency virus [HIV; lymphadenopathy-associated virus (LAV/HTLV- III)] as well as for the presence of cytogenetic abnormalities and monoclonal rearrangements of immunoglobulin and T-cell receptor genes. Both cell lines expressed the same IgM, kappa phenotype as the original lymphoma. The karyotype of Es I was 46, XY, t(8;14), 2 p+, inv (6p), 17p-, and the cells of Es III had an additional i(7q). Immunoglobulin gene studies demonstrated the identical monoclonal rearrangements in both cell lines. Neither EBV nor HIV sequences were detectable in the malignant B cells at the genomic level, leading to the conclusion that mechanisms other than transformation by EBV or HIV may have contributed to the B-cell lymphoma in this patient and possibly also to the generally increased frequency in patients with AIDS.

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

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

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

Rituximab But Not GA101 Is Partially Antagonistic With Inhibitors Of The B-Cell Receptor Pathway In Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4420-4420
Author(s):  
Anna-Katharina Zoellner ◽  
Nico Peter ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Advisory Board ◽  
B Cell Receptor ◽  
Additive Effects ◽  
Anti Cd20 ◽  
The Impact

Introduction Anthracyclin-containing immuno-chemotherapy represents the current standard approach in diffuse large cell B cell lymphoma (DLBCL). However, especially in ABC lymphoma new therapeutic approaches are warranted. Small molecule inhibitors of the B- cell receptor pathway have recently achieved high response rates in several lymphoma subtypes. Since rituximab has been previously described to influence the PI3K- AKT pathway, we investigated the impact of rituximab as well as the novel glycoengineered type II anti-CD20 antibody GA101 (obinutuzumab) in combination with the PI3K- delta inhibitor idelalisib and BTK inhibitor ibrutinib. Methods Established DLBCL ABC (U2932, OCI-Ly10, OCI-Ly3, HBL-1) and GCB (HT, WILL-2, SU-DHL-5, SU-DHL-4, ULA) cell lines were cultivated under standard conditions and exposed to previously determined doses of compounds (rituximab [R]: 1 µg/ml, GA101 [G]: 1 µg/ml, idelalisib [ID]: 5 µM, ibrutinib [I]: 5 nM). Viable cells were determined after 24, 48 and 72 hours based on trypane blue exclusion test. Western blot analysis was performed after 1, 6, 12 and 24 h. All experiments were performed at least in triplicates. Results Rituximab in combination with idelalisib showed differential effects in ABC and GCB cell lines. In ABC cell lines the combination was not superior to single substances after 48 h (OCI-LY10: R: 70%, ID: 83%, R+ID: 76%; U2932: R: 63%, ID: 88%, R+ID: 58%) whereas after 72 h additive effects were observed (OCI-LY10: R: 78%, ID: 77%, R+ID: 57%; U2932: R: 58%, ID: 87%, R+ID: 47%). In GCB cell lines, rituximab and idelalisib again were partially antagonistic and did not increase the effect of single drugs (48 h: ULA: R: 79%, ID: 76%, R+ID: 76%; 72 h: SU-DHL-5: R: 87%, ID: 69%, R+ID: 64%). Combination treatment with GA101 and idelalisib was more effective in both subtypes. In ABC cell lines cell counts were additively reduced after 72 h (U2932: G: 40%, ID: 47%, G+ID: 33%; HBL-1: G: 83%, ID: 86%, G+ID: 63%). Similar additive effects were detected in GCB cell lines (SU-DHL-5: G: 91%, ID: 69%, G+ID: 52%; ULA: G: 59%, ID: 68%, G+ID: 50%). As expected, ibrutinib was not effective in GCB cell lines. In ABC cell lines effects of the combination with rituximab or GA101 were comparable to ibrutinib only (OCI-LY10: R: 84%, I: 51%, R+I: 49%; HBL-1: G: 76%, I: 76%, G+I: 77%). In contrast to published data downregulation of p-AKT was detected after antibody treatment in neither ABC nor GCB cell lines. Idelalisib significantly reduced expression of p-AKT already after 1 h in GCB cell lines (ULA). The combination of idelalisib and GA101 also downregulated potently p-AKT whereas the rituximab combination did not reduce p-AKT expression as pronounced. Similar differences were observed In the ABC cell line U2932. Conclusion The combination of rituximab and idelalisib induced a partially antagonistic effect in GCB cell lines. In contrast, in ABC an additive effect of the combination was observed at all time points. Combination of GA101 and idelalisib was more effective in ABC and GCB lymphoma cell lines potentially due to the more pronounced down regulation of p-AKT. These in vitro data suggest that GA101 may overcome the previously reported antagonism of anti CD20 antibodies and inhibitors of the B-cell receptor pathway. However, the relevance of these data has to be validated in clinical trials. Disclosures: Hiddemann: Hoffmann-La Roche: Support of IITs, Scientiffic advisory board, Speakers honoraria Other. Dreyling:Hoffmann-La Roche: Support of IITs, Speakers honoraria, Support of IITs, Speakers honoraria Other; Janssen: Support of IITs, Scientiffic advisory board, Speakers honoraria Other.

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