B Cell Lymphomas Escape Bone Morphogenetic Protein (BMP) Induced Growth Inhibition: Downregulation of BMP Receptors and Upregulation of Inhibitory Smads.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2605-2605 ◽  
Author(s):  
Kanutte Huse ◽  
Maren Bakkebo ◽  
Morten Oksvold ◽  
Erlend B. Smeland ◽  
June H. Myklebust
Keyword(s):  
B Cells ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
Lymphoma Cell ◽  
Receptor Expression ◽  
Type I ◽  
B Cell Lymphomas

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and mediate their effects mainly through the Smad signaling pathway. TGF-β is one of the most potent negative regulators in hematopoietic cells, and many cancers develop reduced sensitivity towards TGF-β induced growth inhibition by several mechanisms, including functional loss of TGF-β receptors and Smad proteins. We have previously shown that BMP-6 inhibits the growth of normal peripheral blood B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that reduced sensitivity towards BMP-induced growth inhibition might contribute to lymphomagenesis. In the current study, 10 B lymphoma cell lines (representing Burkitt, DLBCL and FL) and tumor material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We found that 5 – 7 out of 10 lymphoma cell lines were resistant towards BMP-2, -4, -6 and -7 induced growth inhibition. In comparison, only 3 of the cell lines were resistant towards TGF-β. Analysis of BMP receptor expression by FACS analysis showed that all lymphoma cell lines and the malignant B cells from primary lymphoma biopsies expressed the BMPR type I Alk-2, whereas the expression of Alk-3 and Alk-6 was variable. Interestingly, the expression of BMPRII was low or undetectable in BMP-6 resistant cell lines, whereas it was highly expressed in 3 out of 4 sensitive cell lines. Also, malignant B cells from lymphoma biopsies showed reduced levels of BMPRII, suggesting that downregulation of BMPRII is a mechanism for evading BMP induced growth inhibition. Interestingly, upregulation of Smad6 or Smad7 was seen in 3 of the BMP-6 resistant cell lines and might represent another way of escaping the inhibitory effects of BMP. The lymphoma cell lines were investigated for endogenous production of BMPs by real-time RT-PCR, and 2 out of 10 cell lines had detectable BMP-6 mRNA, whereas 7 cell lines expressed BMP-7 mRNA. Analysis of purified malignant B cells or normal tumor infiltrating T cells from patient biopsies, confirmed the expression of BMP-6 and BMP-7 in the malignant B cells. Altogether, the data suggest that escape from BMP induced growth inhibition might contribute to increased tumor growth in B cell lymphomas.

Micro-RNA Gene Deregulation by Chromosome Translocations with Fragile Genomic Regions in B-Cell Lymphoma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 786-786
Author(s):  
Bjoern Schneider ◽  
Stefan Nagel ◽  
Maren Kaufmann ◽  
Hans G. Drexler ◽  
Roderick A.F. MacLeod
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Micro Rna ◽  
Lymphoma Cell ◽  
Chromosome Rearrangements ◽  
Causal Connection

Abstract Micro-RNA (miR) genes posttranscriptionally modulate target gene expression via imperfect 3′-UTR matching sequences and play key roles in development, homeostasis and cancer. Little is known how miR genes are themselves regulated, or deregulated in cancer. Chief paradigm for neoplastic miR deregulation concerns miR-17/92 cluster members subject to genomic amplification in B-cell lymphoma. While the repeated occurrence of oncogenic miR genes at or near chromosomal breakpoints in cancer links chromosome fragility to oncogenic miR deregulation, direct evidence of a causal connection remains tenuous. We found that t(3;7)(q27;q32) in a B-cell lymphoma cell line joins 5′-BCL6 to a noncoding region of chromosome 7 inside a common chromosomal fragile site (FRA7H). In these cells hybrid mRNA was absent, unlike canonical BCL6 translocations which involve promoter exchange yielding hybrid mRNA. Affected cells instead showed downregulation of miR-29b-1, the only gene located within FRA7H - a recurrent transcriptional feature of B-cell lymphoma subsets. In another BCL6 translocation, t(3;13)(q27;q31)t(13;12)(q31;p11), which 5′-RACE also showed to be non-fusogenic, long distance inverse (LDI)-PCR revealed junction of 5′-BCL6 to chromosome 13 sequences inside the miR-17/92 host gene MIRH1 (alias c13orf25). FISH using a sensitive tyramide amplification protocol with c13orf25 clones confirmed the presence of a cryptic BCL6-MIRH1 rearrangement. Surprisingly, reverse transcriptase quantitative (q) PCR assay revealed weak MIRH1 expression using 3′-primers. In contrast, repeating the assay using more central primers covering the miR-17/92 coding region showed massive upregulation. 3′-RACE confirmed a novel high level MIRH1 transcript truncated by 3.1 kbp. Quantitative genomic PCR and FISH excluded miR-17/92 genomic copy number alteration, while LDI-PCR analysis showed that formation of truncated MIRH1 involved multiple DNA cuts at 3q27 (x1), 12p11 (x1), and 13q31 (x5) – the last including a complex excision/inversion/insertion rearrangement. Stress induced DNA duplex destabilization (SIDD) analysis revealed that 6 of 7 breaks precisely coincided with fragility peaks. Taken together, these data suggest a novel role for BCL6 translocations in the deregulation of miR genes near sites of chromosome or DNA instability. BCL6 has been shown to suppress p53 in germinal center B-cells thus protecting B-cells from apoptosis induced by DNA damage, offering a possible explanation for chromosome rearrangements associated with genomic fragility therein. Chromosomal MIRH1 dysregulation is not limited to BCL6 expressing lymphomas, however: cytogenetic investigations performed on diverse leukemia-lymphoma cell lines, including those derived from multiple myeloma and plasma cell leukemia, showed 11/50 with cytogenetic rearrangements at or near MIRH1. In sister cell lines sequentially established at diagnosis and relapse of multiple myeloma, only the latter showed miR-17/92 chromosomal rearrangement and upregulation. Interestingly miR overexpression was limited to miR-92, while miR-17/18 were barely expressed. FISH analysis and qPCR showed that discrepant expression was associated with rearrangement upstream of MIRH1. In brief, our data show that like other cancer genes, oncogenic miRs are subject to dysregulation mediated by structural chromosome rearrangements.

B-Cell Receptor Signaling Suppresses TAp63-Induced Apoptosis Via PI3-K/mTOR Pathway and Upregulation of MiR-21 in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 562-562
Author(s):  
Leigh Ann Humphries ◽  
Darcy Bates ◽  
Claire Godbersen ◽  
Prabhjot Kaur ◽  
Alexey V. Danilov
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Mtor Pathway ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Raji Cells ◽  
Transcript Levels

Abstract Abstract 562 p63, an ancestral homolog of p53, encodes two major variants that have variable expression and context-specific functions in malignant tissues. We and others have shown that N-terminally truncated ΔNp63 promotes tumor growth in carcinomas. Meanwhile, the full-length TAp63 variant, which predominates in lymphoid malignancies, is anti-oncogenic in solid tumor models, where it mediates Ras-induced cellular senescence, suppresses anchorage-independent growth, and induces apoptosis. In hepatoma cells, TAp63 activates both extrinsic and intrinsic apoptosis pathways and enhances chemosensitivity. CLL clonal B cells have a low proliferative potential and disrupted apoptotic mechanism as a result of intrinsic defects and interaction with the microenvironment. At the crossroads of those pathways, the B-cell receptor (BCR) serves as a key survival molecule in CLL. Little is known about whether p63 regulates B-cell survival in CLL. Here we sought to investigate the role of TAp63 in regulation of apoptosis in CLL B cells and lymphoma cell lines and determined whether B-cell receptor signaling modulates p63. Forty-eight patients with B-CLL were enrolled in this study. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and co-cultured with M210B4 murine stroma cell line layers in RPMI supplemented with 15% fetal bovine serum (FBS). B-cell lymphoma cell lines Daudi, DOHH, Raji, OCI-LY3, OCI-LY19, SU DHL-4 and SUDHL-10 were maintained in RPMI with 10% FBS. CLL B cells and Raji cells were transfected with TAp63α expression vector or with siRNAs targeting p63 or miR-21 using Lonza Nucleofector with B-cell nucleofection solution (CLL B cells) and Solution V (Raji cells). Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry. B-cell receptor was engaged with 5 mg/mL (Raji cells) or 10 mg/mL IgM (CLL B cells). Expression of p63 and miR-21 was evaluated by immunoblotting and real-time RT-PCR. Median age of patients was 63 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (80%). TAp63α was the predominantly expressed p63 variant in CLL cells and 7 lymphoma cell lines. Compared with normal B cells, TAp63 mRNA transcript levels were low in 28 CLL patient samples (using an arbitrary cutoff of <10% normal; 58.3%) and normal/elevated in 20 samples (41.7%). Patients with high expression of p63 were more likely to exhibit unmutated IGHV (U-CLL; p=0.016). siRNA-mediated knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis of CLL cells cultured on M210B4 murine bone marrow stroma (p<0.01) and was accompanied by a reduction in Noxa, Puma and Bax transcript levels. By contrast, enforced expression of TAp63α enhanced apoptosis. p63 knockdown in the Raji lymphoma cells resulted in increased proliferation and metabolic activity (p<0.05). B-cell receptor engagement suppressed p63 expression in CLL cells and Raji lymphoma cells. Pre-incubation of Raji cells with Syk inhibitor R406 and inhibitors of the PI-3K/mTOR pathway (LY294002, rapamycin, and CAL-101) resulted in the reversal of this phenomenon. Meanwhile, chemical inhibition of MEK, Erk, JNK, and p38 and siRNA-mediated knockdown of the transcription factor FOXO (a downstream targets of PI-3K) had no effect on p63 expression. Since TAp63α is a known target of miR-21, a microRNA that has been implicated in the pathogenesis of CLL, we examined their relationship in CLL and lymphoma. We found that TAp63 transcript levels inversely correlated with the expression of miR-21 in CLL B cells, but not in lymphoma cell lines. BCR stimulation led to increased miR-21 levels in CLL B cells, whereas knockdown of miR-21 resulted in upregulation of TAp63 in 3 out of 5 tested samples. TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells and B-cell lymphoma cell lines, where it modulates the apoptosis program. BCR signaling repressed TAp63α via PI-3K/mTOR pathway and via upregulation of miR-21. This may be particularly relevant in U-CLL, where baseline p63 levels were higher. These data provide additional insights and rationale for targeting the BCR pathway and miR-21 in CLL and lymphoma. Disclosures: No relevant conflicts of interest to declare.

RAD001 (everolimus) down-regulates cyclin D3 and c-Myc and is particularly effective in the treatment of aggressive B-cell lymphomas

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 17573-17573
Author(s):  
S. H. Kuo ◽  
C. H. Hsu ◽  
P. Y. Yeh ◽  
H. C. Hsu ◽  
M. Gao ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Cyclin D3 ◽  
Akt Signaling Pathway ◽  
P70s6 Kinase ◽  
Aggressive B Cell Lymphoma

17573 Background: Aggressive B-cell lymphoma is recently found to be associated with constitutive activation of the PI3K/Akt pathway, and thereby is relatively resistant to apoptosis. Furthermore, activation of the PI3K/Akt signaling pathway can result in the upregulation of cyclin D3 and c-Myc through inhibition of the cap-independent RNA translation. Since mTOR is closely associated with the regulation of the translation process, and is a downstream mediator of the PI3K/Akt signalling pathway, this study aimed to examine if RAD001, an inhibitor of mTOR, can be effective in the treatment of aggressive B-cell lymphoma. Methods: Pfeiffer (diffuse large cell lymphoma), Ramos (EBV (−) Burkitt’s lymphoma), Raji (EBV (+) Burkitt’s lymphoma), and MC116 (EBV (−) undifferentiated lymphoma) cell lines were used in this study. Expression of pAkt, p70S6 kinase, pp70 S6 kinase, 4E-BP1, p4E-BP1, cyclin D3, and c-Myc was measured by immunoblotting. Results: Akt was constitutively activated in all four lymphoma cell lines. Downstream effectors of PI3/Akt signaling pathway, including p70S6 kinase and 4E-BP1, were also phosphorylated in these lymphoma cell lines. RAD001 downregulated p70S6 kinase and 4E-BP1, and the IC50s of growth suppression ranged from 5 to 50 nM, without significant difference between EBV (+) and EBV (−) cell lines. The IC50s of these lymphoma cell lines appeared to be much lower than those obtained from the carcinoma cell lines, suggesting that lymphomas may be particularly sensitive to growth inhibition by RAD001. RAD001 blocked cell cycle progression in G0/G1 phase in all four lymphoma cell lines while there was no significant increase in sub-G1 phases, suggesting that apoptosis was not the major mechanism of RAD001-induced growth inhibition. In addition, in parallel with the RAD001-induced growth inhibition, a dose-dependent down-regulation of the expression of cyclin D3 and c-Myc was demonstrated. Conclusions: The mechanism of action is at least partly due to downregulation of cyclin D3 and c-Myc, and subsequent G0/G1 block. Since overexpression of cyclin D3 and c-Myc is also closely associated with chemoresistance of aggressive B-cell lymphoma, RAD001 may be a useful adjunct in the treatment of this group of tumors. No significant financial relationships to disclose.

B Cell Lymphomas Are Resistant towards Bone Morphogenetic Protein (BMP) Induced Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2381-2381
Author(s):  
Kanutte Huse ◽  
Marianne B. Eide ◽  
Christian Kersten ◽  
Erlend B. Smeland ◽  
June H. Myklebust
Keyword(s):  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Bmp Receptors ◽  
Induced Apoptosis

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily, and mediate their effects mainly through the Smad signalling pathway. Whereas TGF-β is well established as one of the most potent negative regulators in hematopoietic cells, the role of BMPs remains more elusive. We have previously shown that BMP-6 inhibits the growth of naïve and memory human B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that resistance towards BMP-induced growth inhibition is a possible mechanism for lymphomagenesis. In the current study, 7 B cell lymphoma cell lines (representing Burkitt lymphoma (BL) and DLBCL) and tumour material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We analyzed the expression of BMP receptors by FACS analysis, and found variable expression of the BMP receptor type I (Alk2, Alk3 and Alk6) and type II (BMP RII, Activin RIIA and RIIB) among the cell lines and in primary lymphoma cells, suggesting variable binding of BMPs. We next investigated the effect of BMP-2, BMP-4, BMP-6 and BMP-7 on proliferation and survival of B lymphoma cell lines, and found 2 of 7 cell lines to be resistant towards BMP-2 and BMP-4 induced growth inhibition. In contrast, 4 of 7 and 7 of 7 cell lines were resistant to BMP-6 and BMP-7 induced growth inhibition, respectively. In Sudhl6 cells that were highly sensitive to BMP-2 and BMP-6 induced apoptosis and inhibition of proliferation, we demonstrated that the cytokines IL-10, CD40 Ligand and BLyS were able to counteract the negative effects induced by BMPs, while IL-2 and IL-4 were not. On the contrary, both BMP-2 and BMP-6 greatly increased anti-IgM activation induced apoptosis. In resistant lymphoma cells, the BMPs were not able to induce detectable levels or induced low levels of phosphorylated SMAD1/5/8 compared to sensitive cell lines. Low or no increase in phosphorylation of SMAD1/5/8 induced by BMPs could only partly be explained by low/ undetectable expression of BMP receptors. Hence, upregulation of inhibitory Smads (Smad6, Smad7) or mutations in receptors or Smads represent other possible mechanisms for resistance to BMPs in lymphomas, and this is currently under investigation. We also investigated if the lymphoma cells produced BMPs themselves and found that 5 of 7 cell lines and 3 of 5 primary lymphomas produced significant amounts of BMP-7. Some lymphoma cells also had detectable levels of BMP-4 and BMP-6. Our findings that lymphoma cells are resistant towards BMP-7 and to some degree BMP-6 induced growth inhibition, whereas they produce these cytokines, suggest that resistance towards BMP induced signalling in B cell lymphomas can contribute to increased tumour growth.

GX15-070 and Bortezomib Induce Up-Regulation of BH3 Single Domain Pro-Apoptotic Proteins Puma and Noxa and Is Associated with Synergistic Anti-Tumor Activity in Rituximab-Sensitive, Rituximab-Resistant Cell Lines (RSCL and RRCL), and Primary Lymphoma Patient Specimens.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1389-1389 ◽  
Author(s):  
Elizabeth A. Gruber ◽  
Myron S. Czuczman ◽  
Scott H. Olejniczak ◽  
Joy Knight ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
Single Domain ◽  
Primary Lymphoma ◽  
B Cell Lymphomas ◽  
Bh3 Domain ◽  
In Vitro Exposure

Abstract The interactions between the members of the BH3 domain family of proteins play an important role in the development, progression, and prognosis in various subtypes of B-cell lymphomas. Therapies that selectively favor a pro-apoptotic environment are attractive strategies to overcome chemotherapy resistance in B-cell lymphomas. We previously reported that by targeting Bcl-2 family proteins with either Bcl-2 anti-sense oligonucleotides or GX15-070, a novel pan-inhibitor of the Bcl-2 family members, improved rituximab and/or chemotherapy activity in vitro and/or in vivo (Ramanarayanan J, et al, BJH2004; 127:519–30 and Hernandez-Ilizaliturri F, et al, Blood2006; 108:2502a/2523a). Recently, investigators have demonstrated that the proteasome is an important regulator of various members of Bcl-2 family proteins. In our efforts to increase the therapeutic options for B-cell lymphoma patients we studied the biological effects of GX15-070 in combination with the proteosome inhibitor bortezomib in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL, or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. In addition, we utilized lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed diffuse large B-cell lymphomas (DLBCL). NHL cells were exposed in vitro to escalating doses of GX15-070 (0, 2, 5, 10 and 20μM) and/or Bortezomib (0, 2, 10 and 20nM) for 24 and 48 hrs. Changes in the expression of BH3 domain Bcl-2 proteins were studied by Western blot (i.e. Bcl-2, Mcl-1, Bcl-XL, Bad, Bid, Bax, Bak, Puma, and Noxa). NHL cell lines and DLBCL cells isolated from patients were exposed to different concentrations of GX15-070 (0 to 20μM) with or without Bortezomib (0 to 20nM). Cell viability was determined by the Cell Titer-Glow luminescent assay, and DNA synthesis was evaluated by standard [3H]-Thymidine incorporation assays at 24 and 48 hrs. Statistical differences were analyzed by Chi-square test. In vitro exposure of RRCL and RSCL to GX15-070 resulted in a significant up-regulation of Puma while in vitro exposure of the same cells to bortezomib led to a dose- and time-dependent up-regulation of Noxa and Bak. In vitro exposure of RRCL, RSCL, and primary lymphoma specimens to GX15-070 and bortezomib resulted in significant synergistic activity compared to controls. In summary, deregulation of apoptosis by BH3 inhibition with GX15-070 and bortezomib: induces the expression of BH3 single domain proteins Puma and Noxa; results in cell death and antiproliferation not only in RSCL and RRCL, but also from “treatment-refractory” primary DLBCL patient samples. Our findings strongly suggest that GX15-070 added to bortezomib may result in a novel and potent therapeutic strategy against aggressive B-cell lymphomas.

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

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

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

scholarly journals Inhibiting the Nuclear Exporter XPO1 and the Antiapoptotic Factor BCL2 Is Synergistic in XPO1 Mutant and Wildtype Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Frank Owusu-Ansah ◽  
Jumana Afaghani ◽  
Stanley Lee ◽  
Justin Taylor
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
Mutant Cell ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
B Cell Lymphomas ◽  
Increased Sensitivity ◽  
Refractory Lymphoma

We have recently shown that XPO1 mutations are drivers of lymphomagenesis and occur across B-cell lymphomas, specifically in chronic lymphocytic leukemia (CLL), classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. The co-occurrence of other oncogenic events cooperating with XPO1 provides an opportunity for combined targeted therapy. Increased expression of the anti-apoptotic factor BCL2 has long been known to be a critical part of the pathophysiology of B-cell lymphomas. Recently, the oral BCL2 inhibitor, venetoclax, was approved in CLL and is currently being evaluated in clinical trials for other B-cell lymphomas. Selinexor is a potent, oral XPO1 inhibitor that was recently approved in multiple myeloma and diffuse large B-cell lymphoma. XPO1 inhibition exerts its antineoplastic effects by blocking key lymphomagenic pathways, such as NFκB, and decreasing the anti-apoptotic protein survivin. We therefore hypothesized that combining selinexor and venetoclax would have potential synergy and provide an oral precision combination therapy for relapsed, refractory lymphoma. We first set out to determine whether XPO1 mutant lymphoma cell lines showed differential response to either selinexor or venetoclax monotherapy. Five lymphoma cell lines; 3 diffuse large B-cell lymphoma (SU-DHL-6, SU-DHL-16, FARAGE) and 2 classical Hodgkin lymphoma (L428 and SUP-HD1), were subjected to next-generation sequencing (NGS) to assess for the presence or absence of XPO1 mutations. SUDHL-16 and SUP-HD1 were heterozygous for the XPO1 E571K hotspot mutation while SUDHL-6, FARAGE and SUP-HD1 were wildtype at the XPO1 locus. These 5 cell lines were used to assess sensitivity to Selinexor and/or Venetoclax. The CellTiter Glo assay was used to assess cell viability after 72 hours of treatment. Assays were performed in triplicate on 96-well plates that were read using a Spectramax plate reader. The XPO1 mutant cells showed increased sensitivity to selinexor (XPO1 mutant IC50 = 16-35nM; XPO1 WT IC50 = 41-231nM) as previously seen in conditional knockin mouse models of XPO1 mutant CLL (Figure A). Additionally, the XPO1 mutant cell lines showed increased sensitivity to single-agent venetoclax (XPO1 mutant IC50 = 2-13nM; XPO1 WT IC50 = 5-2853nM), an observation that has not previously been made (Figure B). Next, we tested the synergy of the combination of selinexor and venetoclax in the XPO1 mutant and wildtype cell lines. Increasing concentrations of the individual drugs were applied to each individual cell line in a 6x6 matrix. The cell viability percentage for each concentration was then entered into a synergy finder (www. synergyfinder.fimm.fi). The Bliss Independence model was used to calculate synergy of the Selinexor-Venetoclax combinations. As hypothesized, the combination of selinexor and venetoclax indeed showed synergy in both the wildtype and mutant XPO1 cell lines. Furthermore, the XPO1 mutant cell lines showed a higher degree of synergy compared to the wildtype cells (Figure C). Finally, a remarkable patient allowed us to test this combination ex vivo. This patient with CLL had undergone multiple therapies including chemoimmunotherapy, ibrutinib and venetoclax monotherapies. This patient had a founder XPO1 E571K mutation and also had acquired a BTK C481S ibrutinib resistance mutation and MYC amplification. These cells were unique in that they were easily able to be tested in ex-vivo culture to test sensitivity to different therapies. When tested with chemotherapy or ibrutinib they were completely resistant, and even with venetoclax they were fairly resistant; however, they remained sensitive to XPO1 inhibition with Selinexor. Selinexor and venetoclax showed remarkable synergy measured by a BLISS delta score of 18.78 (Figure D). In conclusion, inhibiting the nuclear exporter XPO1 and the anti-apoptotic factor BCL2 is synergistic in both XPO1 wildtype and mutant lymphoma. XPO1 mutant lymphomas show increased sensitivity to both selinexor and venetoclax. Additionally, selinexor and venetoclax showed a higher degree of synergism in XPO1 mutant lymphoma cell lines and were highly synergistic in primary XPO1 mutant CLL patient cells ex vivo. This combination is highly promising as an all oral alternative for relapsed, refractory lymphoma. Next steps include preclinical testing in mouse models in vivo using XPO1 mutant and wildtype mice crossed with mice overexpressing BCL2. Figure Disclosures No relevant conflicts of interest to declare.

Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1401-1401 ◽  
Author(s):  
Massimo Mangiola ◽  
Kate Welsh ◽  
Shinichi Kitada ◽  
Irene M. Pedersen ◽  
Nuzhat Pathan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
Efficient Induction ◽  
Anti Cd20

Abstract We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

Inactivation Of BANK1 By a Novel IGH-Associated Translocation and 5’ Hypermethylation In B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2497-2497 ◽  
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Jiong Yan ◽  
Leonardo Boiocchi ◽  
Shuhua Cheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Methylation Status ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Intron 1 ◽  
Large B Cell

Abstract A novel IGH-associated reciprocal translocation, t(4;14)(q24;q32), was identified, along with trisomy 9, in 20 of 20 metaphases by conventional karyotyping in a case of malignant gastric post-transplant lymphoproliferative disorder (PTLD). Cloning of the translocation site by inverse PCR identified BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific adaptor protein with putative functions in B-cell receptor and CD40 signaling, as a novel IGH translocation partner. The breakpoints were located at the Sα region of IGH and intron 1 of BANK1. The translocation juxtaposed the two genes in opposite orientations, and surprisingly, resulted in transcriptional inactivation of BANK1 as a result of dissociation of the major BANK1 promoter. While BANK1 isoforms were expressed in all tonsillar B-cells, with lower levels (∼ 5 fold) in the germinal centers (GC) compared to naïve and memory B-cells, transcription from the major promoter in the tumor was absent and transcription from the minor promoter was reduced 50% relative to GC B-cells, suggesting that the non-translocated BANK1 allele was also inactivated. The total BANK1 expression was very low (∼10% of normal GC B cells) and crytic promoter activation was not identified. Several genes (PPP3CA, MIR1255A, FLJ20021 and SLC39A8), located 180 to 440 kb away from BANK1, were analyzed for mRNA expression; there is no significant activation in any of these genes, further supporting that BANK1is indeed the target gene affected by the translocation. Interphase FISH using break-apart BANK1 probes confirmed breakpoint in the index case but did not identify translocations in additional 15 PTLDs and 68 diffuse large B-cell lymphomas (DLBCL), implying that BANK1 translocation may be a rare event. To determine if BANK1 inactivation may occur in B-cell lymphomas by other mechanisms, 23 B-cell lymphoma cell lines, including 8 Burkitt lymphoma (BL), 9 diffuse large B cell lymphoma (DLBCL), 3 primary effusion lymphoma (PEL), and 3 classical Hodgkin lymphoma (cHL) were bisulfite sequenced to assess the methylation status of 37 CpG dinucleotides in a 436 base-pair region at the 5’ end of BANK1, which extends across exon 1 into the 5’ portion of intron 1. High level of methylation (>60% methylation on average among all CpGs) was seen in all 3 cHL and 2 of 3 PEL cell lines. Regional methylation was seen in 3 of 8 BL lines and 1 of 3 PEL lines. No hypermemethylation was identified in the DLBCL lines or in normal tonsils. Hypermethylation was associated with almost complete silencing of BANK1 transcription. In the DLBCL lines and BL lines without BANK1 hypermethylation, BANK1mRNA expressions were variable, ranging from <5% to 130% of GCB cells. To confirm that BANK1 hypermethylation is present in primary lymphoma cases, methylation status of 17 of the 37 CpGs were assessed in 23 cHL cases using en bloc formalin-fixed, paraffin-embedded materials and also laser-capture micro-issected Hodgkin/Reed-Sternberg (HRS) cells. There was evidence of BANK1 hypermethylation in the tumor cells in 9 of 23 cHL. Tumor cell specificity of BANK1 hypermethylation was further confirmed in 4 cHL cases using micro-dissected HRS cells. HRS cells were negative for BANK1 in 28 of 29 cHL cases examined by immunohistochemistry, suggesting that other mechanisms other than DNA methylation may be responsible for silencing BANK1expression. To investigate whether BANK1 has biological effects on B-cells related to lymphoma development, exogenous BANK1 was re-introduced to BC3, a PEL cell line showing marked BANK1 hypermethylation with absence of BANK1 expression. We established a stable doxycycline-inducible BC3 cell line expressing BANK1. Inhibition of cell growth was observed 2 to 3 days after doxycyline induction, and the number of viable cells with transfected BANK1 was only 25% compared to BC3 cells carry vehicle alone at day 6. An analysis of 5-bromo-2’ deoxyuridine (BrdU) incorporation after 48 hours of doxycline induction revealed that the fraction of cells in S-phase was reduced by 50% in the BANK1 transfectants, suggesting that BANK1has a negative effect on cell proliferation in these B cells. In summary, we have identified a novel IGH translocation partner and provide an example of an unusual consequence (gene inactivation) of IGH-associated translocation. We provide for the first time evidence of a potential role of BANK1 down-regulation in the development of B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prosurvival and Chemoprotective Effects of Tumor-Associated Macrophages Reversed By Anti-CSF-1R Monoclonal Antibody in B-Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 498-498
Author(s):  
Anupama Gopisetty ◽  
Myriam Foglietta ◽  
Min Zhang ◽  
Zhiqiang Wang ◽  
Nathan Fowler ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Tumor Survival

Abstract The results of gene expression profiling (GEP) and immunohistochemical studies indicate that survival is worsened by macrophages (MΦ) in the tumor microenvironment of various B-cell lymphomas including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Tumor-associated macrophages (TAMs) are known to be different from other types of MΦ, but the effects of TAMs that worsen prognosis in B-cell lymphoma are essentially unknown, as are the mechanisms of these effects. Here, we determined the phenotype and effects of TAMs on tumor survival, proliferation, and drug resistance in B-cell lymphomas and evaluated strategies to reverse their effects. As compared to peripheral blood monocytes (Mo) from normal donors (ND), Mo from FL patients were differentiated less into M1 MΦ (defined as CD68+CD163loCD206loCD86hi) by culture with CSF-1 for 5 days followed by IFN-g + LPS for 2 days more. In contrast, Mo from FL patients and ND were differentiated similarly into M2 MΦ (defined as CD68+CD163hiCD206hiCD86lo) by culture with CSF-1 followed by IL-4. Consistent with this, MΦ gene signatures from FL tumors were more similar to previously-described signatures of M2 rather than M1 MΦ (Martinez et al, J Immunol, 2006, 177(10):7303-11). In co-culture, primary FL tumor cells and lymphoma cell lines (including RL, a transformed FL cell line; Granta 519, a mantle cell lymphoma (MCL) cell line; and Raji, a Burkitt lymphoma cell line) induced differentiation of Mo into MΦ. Differentiation could be prevented by CS4 monoclonal antibody (mAb), a fully human IgG1 anti-human CSF-1R mAb (ImClone/Eli Lilly), but not isotype control Ab. Elevated levels of CSF-1 in culture supernatants after addition of CS4 mAb and real-time PCR of tumor cells suggested secretion of CSF-1 by lymphoma cells. Spontaneous apoptosis of primary FL and MCL tumor cells, determined by Annexin V and propidium iodide staining, was significantly reduced by co-culture with ND Mo (p<0.01), whether pre-differentiated into MΦ with CSF-1 or not, but this protection could be reversed by CS4 mAb. Mo and/or pre-differentiated MΦ protected primary FL and MCL tumor cells from cytotoxic effects of doxorubicin and/or bendamustine (p<0.01), but CS4 mAb reversed this effect. To assess effects of MΦ on proliferation, lymphoma cell lines (RL, Granta 519, and Raji) were CFSE-labeled prior to co-culture with Mo and doxorubicin, and proliferation assessed by CFSE dilution by flow cytometry in the presence or absence of CS4 or isotype control mAbs. MΦ promoted proliferation of all three cell lines, but this effect could be reversed by CS4 mAb. To further understand the mechanism by which MΦ promote tumor survival and growth, we performed phosflow analysis and found increased phosphorylation of STAT3 in co-cultured lymphoma cells. Consistent with this, we observed a correlation between an 11-gene STAT3 activation signature, described by Huang et al in DLBCL tumors (J Clin Oncol, 2013, 52.8414), and a MΦ gene signature in whole genome GEP studies of 191 FL tumors (Pearson correlation co-efficient=0.396, p<0.001). In conclusion, our results suggest that Mo from FL patients are predisposed to differentiate into an M2-like MΦ state. The interaction between lymphoma cells and Mo/MΦ is reciprocal: a change in Mo (MΦ differentiation) induced by interaction with lymphoma tumor cells leads to a change in the tumor cells (promotion of survival, proliferation, and chemoresistance). More importantly, our results demonstrate that targeting TAMs using CS4, an anti-CSF-1R mAb, can be an effective strategy to overcome the adverse effects of TAMs and reverse chemoresistance. Further studies are needed to determine whether STAT3 activation contributes to the protumor effects of TAMs. This may provide novel insights into the molecular mechanisms related to TAMs and lymphoma cells and offers additional targets for therapeutic development. In the long term, strategies targeting TAMs is especially appealing, as they should be able to be combined with existing therapies including chemotherapy, other immunotherapy, and targeted therapy, potentially improving their efficacy without increasing toxicity for FL, DLBCL, and other B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

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