Hypermethylation of CD44 Is Characteristic of Specific Lymphoma Subtypes.

Abstract Abstract 3469 Poster Board III-357 Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG island hypermethylation profiles are specific for different tumor types [Costello et al., 2000, Nat Genet 25:132-138]. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes. Disclosures No relevant conflicts of interest to declare.

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Long Non-Coding RNAs As Components Of The MYC Network In B Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.1260.1260 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1260-1260

Author(s):

Joost Kluiver ◽

Melanie Winkle ◽

Mina Tayari ◽

Martijn Terpstra ◽

Gertrud Kortman ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Expression Patterns ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Differentially Expressed ◽

Primary Lymphoma ◽

Aberrant Expression

Abstract MYC is an important oncogenic transcription factor in B-cell lymphoma and high MYC expression is associated with aggressive behavior and poor clinical outcome. A large number of genes are regulated by MYC, several of which are shown to contribute to the MYC induced phenotype. Long non-coding (lnc)RNAs have recently emerged as a novel class of regulatory RNAs acting at the epigenetic, transcriptional or posttranscriptional level. Aberrant expression of several lncRNAs has already been implicated in various aspects of tumorigenesis. It is currently unknown to what extend MYC can regulate lncRNA expression and whether these lncRNAs contribute to the pathogenesis of B-cell lymphoma. Using an inducible MYC B cell lymphoma model and a custom microarray we investigated the expression >10,000 lncRNA loci and identified 1,820 lncRNA probes that show a MYC regulated expression pattern. Of these, 355 responded already after 4h, indicating direct MYC regulation. To identify transcripts relevant to lymphoma pathogenesis, we determined if these 355 lncRNAs were differentially expressed between primary lymphoma cases with high and low MYC expression and in addition also between MYC-high lymphoma cell lines and normal germinal center B cells. This revealed an overlap of 176 lncRNAs that were MYC regulated, aberrantly expressed in B cell lymphomas and differentially expressed between MYC-high and MYC-low lymphomas. Differential expression patterns were validated by qRT-PCR. As a first indication for lncRNA function, we isolated RNA from nuclear and cytoplasmic fractions of B cell lymphoma cell lines and determined enrichment fold in comparison to RNA isolated from the total cell lysates. Approximately 40% of all lncRNA transcripts showed specific subcellular localization, 80% nuclear and 20% cytoplasmic enriched. 31 of the 176 candidate lncRNAs were enriched in a specific cellular fraction. Furthermore, we analyzed which lncRNAs are enriched in Argonaute 2 containing complexes as an indication for lncRNA-miRNA interaction. For ∼5% of all expressed lncRNAs we found evidence for miRNA-lncRNA interactions, including 8 of the 176 differentially expressed MYC-induced lncRNAs. This study identified 176 MYC responsive lncRNAs that are deregulated in B cell lymphoma. To establish a definitive role in B cell lymphoma pathogenesis a further characterization is warranted. Disclosures: No relevant conflicts of interest to declare.

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Combination of Enzastaurin and HDAC Inhibitors Synergically Induce Apoptosis in Lymphoma Cells.

Blood ◽

10.1182/blood.v114.22.4783.4783 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4783-4783

Author(s):

Juraj Bodo ◽

Jan Sedlak ◽

Jaroslaw P. Maciejewski ◽

Eric D. Hsi

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Parp Cleavage ◽

Primary Lymphoma ◽

Low Concentrations

Abstract Abstract 4783 Introduction Histone deacetylase inhibitors (HDACis) are approved for use in the setting of cutaneous T-cell lymphoma with modest benefit. Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphoma. Specifically, enzastaurin-induced inhibition of PKC leads to rapid accumulation of β-catenin that triggers c-Jun dependent induction of p73, followed by apoptosis. We investigated the cytotoxicity and mechanisms of cell death of combination enzastaurin and low concentrations of HDACis in B-cell lymphoma and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Experimental design Apoptosis was measured by flow cytometry and PARP cleavage. Phospho-GSK3β (S9), pS6, phospho-c-jun (S73) and β-catenin were analyzed by Western blot or quantum-dot immunoflourescence as measures of PKCβ inhibition. Cytotoxicity was determined by WST-1 proliferation assay and colony forming cell (CFC) assays. Results As expected, enzastaurin induced dephosphorylation of GSK3β and S6RP associated with increased β-catenin expression followed by phosphorylation of c-jun (S73) and PARP cleavage in SU-DHL-6 (diffuse large B-cell lymphoma line) cells. Treatment with low concentrations of suberoylanilide hydroxamic acid (SAHA) showed slight or no changes in studied proteins. Combined enzastaurin/SAHA treatment resulted in strong synergistic apoptosis in two treated germinal center B-cell-like and two activated B-cell-like lymphoma cell lines, two T-cell lymphoma cell lines and four different primary lymphoma/leukemia samples. Similarly, combined enzastaurin/ valproic acid treatment induced synergistic apoptosis in SU-DHL-6 cell line, suggesting the synergy is generalizable to other HDACis. In comparison to the single agent treatment, combined enzastaurin/ SAHA treatment resulted in activation of proapoptotic MAPK, c-jun N-terminal kinase, further increase of phospho c-jun (S73) levels, increased FasL levels, and amplification of PARP cleavage. Quantitative immunofluorescence assay showed a more rapid increase of β-catenin levels with the combination than either agent alone. Furthermore, compared to the low dose SAHA treatment alone, hyperacetylation of histone H3 was detected in samples when enzastaurin was added in combination with low dose SAHA, likely the consequence of displacement of HDAC by β-catenin. In addition, no change in CFC output in normal bone marrow exposed to this combination was observed. Conclusion Enzastaurin/ HDACi therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancy through increased biochemical effects attributed to each agent. These data support further investigation of addition of PKCβ inhibitors to HDACi in order to increase their anti-lymphoma effects. Disclosures: No relevant conflicts of interest to declare.

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Targeting Translation Bypasses Pim Kinase Activity, a Common and Adverse Prognostic Marker In Lymphoma

Blood ◽

10.1182/blood.v116.21.119.119 ◽

2010 ◽

Vol 116 (21) ◽

pp. 119-119

Author(s):

Jonathan H. Schatz ◽

Julie Teruya-Feldstein ◽

Man Jiang ◽

Andrew D Zelenetz ◽

Hans-Guido Wendel

Keyword(s):

Follicular Lymphoma ◽

Prognostic Marker ◽

Cell Lines ◽

Kinase Activity ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Phosphorylation Site ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Model Systems

Abstract Abstract 119 The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, but rapamycin-analog (rapalog) mTOR inhibitors have shown only modest benefits in clinical trials on lymphoma patients. To better understand the resistance of lymphomas to rapalogs, we have undertaken a study of the Pim family kinases, which signal in parallel to PI3K/AKT/mTOR and whose expression has been detected in multiple subtypes of non-Hodgkin lymphoma (NHL). The two pathways converge on activation of cap-dependent translation, suggesting new treatment possibilities by targeting this common downstream output. To assess the clinical relevance of Pim activity, we have quantified Pim1 and Pim2 expression in multiple NHL subtypes using tissue microarray (TMA) technology. We find common expression of Pim1, Pim2, or both proteins in diffuse large B-cell lymphoma (DLBCL, 65.5%), follicular lymphoma (58%), small lymphocytic lymphoma (76.5%) and mantel cell lymphoma (89.7%). Importantly, Kaplan-Meier survival analysis of clinical data linked to our DLBCL TMAs show a strong trend toward a worse overall survival when Pim expression is present in diagnostic tumor samples compared to Pim-negative tumors (p=0.0965). Studies in vivo demonstrate Pim's ability to accelerate oncogenesis in a manner similar to AKT in model systems specific to Burkitt's lymphoma (Eμ -Myc) and follicular lymphoma (VavP-Bcl2). Treatment studies in secondary recipient animals show that Pim promotes resistance to anthracycline chemotherapy like AKT. However, Pim tumors completely resist rapamycin in stark contrast to AKT tumors. To elaborate on these findings, we have employed a genetically defined system of rapamycin sensitivity lacking mTOR's upstream repressor TSC2. These studies show that Pim's ability to mediate rapamycin resistance is dependent on its ability to maintain inhibitory phosphorylation of the translation repressor 4EBP1. Specifically, a phosphorylation-site deficient mutant of 4EBP1 completely abrogates Pim's ability to maintain the viability of the TSC2 −/− cells. We have expanded on this finding using the drug silvestrol, which inhibits cap-dependent translation by targeting eIF4A. Silvestrol shows high potency against Pim-expressing TSC2 −/− cells (IC50 < 1 nM) and also against a panel of Pim-expressing lymphoma cell lines (IC50 1–10 nM). Indeed, targeting cap-dependent translation appeared more effective than the Pim kinase inhibitor SGI-1776 (IC50 1–10 μ M against lymphoma cell lines), which has significantly higher potency against Pim1 than Pim2. In conclusion, we have more clearly defined Pim kinase activity as a major mediator of oncogenesis in multiple NHL subtypes and as a likely negative prognostic marker in DLBCL. Our mechanistic and treatment studies provide a strong rational basis for targeting cap-dependent translation as a treatment strategy to bypass Pim activity and improve lymphoma patients' responses to both cytotoxic and rapalog therapies. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.4721.4721 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4721-4721

Author(s):

Bing Xia ◽

Xiaowu Li ◽

Le Zhang ◽

Qing Guo ◽

Xin Jin ◽

...

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Lymphoma Cell ◽

Primary Lymphoma ◽

Lymphoma Cells

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

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Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Blood ◽

10.1182/blood.v122.21.5150.5150 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5150-5150

Author(s):

Indira D Joshi ◽

Mitchell R Smith

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Low Level ◽

Induced Apoptosis

Abstract Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v108.11.234.234 ◽

2006 ◽

Vol 108 (11) ◽

pp. 234-234 ◽

Cited By ~ 1

Author(s):

Tait D. Shanafelt ◽

Yean K. Lee ◽

Susan M. Geyer ◽

Deanna Grote ◽

Mary Stenson ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Green Tea ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Primary Lymphoma ◽

Lymphoma Cells

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

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