Constitutive Activation of Extracellular Signal-Regulated Kinase Predisposes Diffuse Large B-Cell Lymphoma Cell Lines to CD40-Mediated Cell Death

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.

Download Full-text

Vav-1 expression correlates with NFκB activation and CD40-mediated cell death in diffuse large B-cell lymphoma cell lines

Hematological Oncology ◽

10.1002/hon.935 ◽

2010 ◽

pp. n/a-n/a ◽

Cited By ~ 2

Author(s):

Annette Hollmann ◽

Raquel Aloyz ◽

Kristi Baker ◽

Stephan Dirnhofer ◽

Trevor Owens ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Large B Cell Lymphoma ◽

Large B Cell

Download Full-text

Extracellular Signal-Regulated Kinase Positively Regulates the Oncogenic Activity of MCT-1 in Diffuse Large B-Cell Lymphoma

Cancer Research ◽

10.1158/0008-5472.can-09-1606 ◽

2009 ◽

Vol 69 (19) ◽

pp. 7835-7843 ◽

Cited By ~ 19

Author(s):

Bojie Dai ◽

X. Frank Zhao ◽

Patrick Hagner ◽

Paul Shapiro ◽

Krystyna Mazan-Mamczarz ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Extracellular Signal Regulated Kinase ◽

Large B Cell Lymphoma ◽

Extracellular Signal ◽

Oncogenic Activity ◽

Large B Cell

Download Full-text

Upregulation of Cereblon Expression By the DNA Methyltransferase Inhibitor Azacytidine Strongly Enhances Lenalidomide Cytotoxicity in Germinal Center B-Cell-like (GCB) and Activated B-Cell-like (ABC) Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽

10.1182/blood.v124.21.2253.2253 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2253-2253 ◽

Cited By ~ 2

Author(s):

Silvia L Locatelli ◽

Roberto Papait ◽

Giuseppa Careddu ◽

Ada Koschorke ◽

Giuliano G Stirparo ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Germinal Center B Cell ◽

Large B Cell

Abstract INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL. METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment. RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL. CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Abstract 4778: Resveratrol suppresses constitutive activation of AKT via generation of reactive oxygen species and induces apoptosis in diffuse large B cell lymphoma cell lines

10.1158/1538-7445.am2011-4778 ◽

2011 ◽

Author(s):

Zeenath Jehan ◽

Azhar R. Hussain ◽

Maqbool Ahmed ◽

Saeeda O. Ahmed ◽

Fouad Al-Dayel ◽

...

Keyword(s):

Reactive Oxygen Species ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Reactive Oxygen ◽

B Cell Lymphoma ◽

Oxygen Species ◽

Constitutive Activation ◽

Large B Cell Lymphoma ◽

Large B Cell

Download Full-text

Resveratrol Suppresses Constitutive Activation of AKT via Generation of ROS and Induces Apoptosis in Diffuse Large B Cell Lymphoma Cell Lines

PLoS ONE ◽

10.1371/journal.pone.0024703 ◽

2011 ◽

Vol 6 (9) ◽

pp. e24703 ◽

Cited By ~ 51

Author(s):

Azhar R. Hussain ◽

Shahab Uddin ◽

Rong Bu ◽

Omar S. Khan ◽

Saeeda O. Ahmed ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Constitutive Activation ◽

Large B Cell Lymphoma ◽

Large B Cell

Download Full-text

The novel anti-MEK small molecule AZD6244 induces BIM-dependent and AKT-independent apoptosis in diffuse large B-cell lymphoma

Blood ◽

10.1182/blood-2011-03-340109 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1052-1061 ◽

Cited By ~ 24

Author(s):

Savita Bhalla ◽

Andrew M. Evens ◽

Bojie Dai ◽

Sheila Prachand ◽

Leo I. Gordon ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Small Molecule ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

The Novel ◽

Primary Cells ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract The RAS/RAF/MEK/ERK signaling pathway has been largely unexplored as a potential therapeutic target in lymphoma. The novel 2nd generation anti-MEK small molecule, AZD6244, down-regulated its direct downstream target, phospho-ERK (pERK) in germinal center and nongerminal center diffuse large B-cell lymphoma (DLBCL) cell lines and primary cells. Similar decreased pERK levels were noted despite constitutive activation (CA) of MEK. Consequently, several lymphoma-related ERK substrates were down-regulated by AZD6244 including MCT-1, c-Myc, Bcl-2, Mcl-1, and CDK1/2. AZD6244 induced time- and dose-dependent antiproliferation and apoptosis in all DLBCL cell lines and fresh/primary cells (IC50 100nM-300nM). Furthermore, AZD6244 resulted in significantly less tumor compared with control in an in vivo DLBCL SCID xenograft model. Cell death was associated with cleaved PARP, caspases-8, -9, and -3, and apoptosis was caspase-dependent. In addition, there was stabilization of FoxO3a, activation of BIM and PUMA, and a significant decrease in c-Myc transcripts. Moreover, siRNA knockdown of BIM abrogated AZD6244-related apoptosis, while shRNA knockdown of ERK minimally sensitized cells. Finally, manipulation of AKT with transfection of OCI-LY3 cells with CA-AKT or through chemical inhibition (LY294002) had minimal effect on AZD6244-induced cell death. Altogether, these findings show that the novel anti-MEK agent, AZD6244, induced apoptosis in DLBCL and that cell death was BIM-dependent.

Download Full-text

MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

Journal of Oncology ◽

10.1155/2019/9832956 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Danxia Zhu ◽

Cheng Fang ◽

Wenting He ◽

Chen Wu ◽

Xiaodong Li ◽

...

Keyword(s):

B Cell ◽

Binding Site ◽

Cell Lines ◽

Expression Vector ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Protein Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

Download Full-text

Repurposing dasatinib for diffuse large B cell lymphoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905239116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 16981-16986 ◽

Cited By ~ 5

Author(s):

Claudio Scuoppo ◽

Jiguang Wang ◽

Mirjana Persaud ◽

Sandeep K. Mittan ◽

Katia Basso ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Kinase Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Multikinase Inhibitor ◽

Large B Cell Lymphoma ◽

Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

Download Full-text

Genome-wide characterization of copy number variations in diffuse large B-cell lymphoma with implications in targeted therapy

Precision Clinical Medicine ◽

10.1093/pcmedi/pbz024 ◽

2019 ◽

Vol 2 (4) ◽

pp. 246-258

Author(s):

Prashanthi Dharanipragada ◽

Nita Parekh

Keyword(s):

B Cell ◽

Cell Lines ◽

Copy Number ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Copy Number Variations ◽

Model Systems ◽

Significant Heterogeneity ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the aggressive form of haematological malignancies with relapse/refractory in ~ 40% of cases. It mostly develops due to accumulation of various genetic and epigenetic variations that contribute to its aggressiveness. Though large-scale structural alterations have been reported in DLBCL, their functional role in pathogenesis and as potential targets for therapy is not yet well understood. In this study we performed detection and analysis of copy number variations (CNVs) in 11 human DLBCL cell lines (4 activated B-cell–like [ABC] and 7 germinal-centre B-cell–like [GCB]), that serve as model systems for DLBCL cancer cell biology. Significant heterogeneity observed in CNV profiles of these cell lines and poor prognosis associated with ABC subtype indicates the importance of individualized screening for diagnostic and prognostic targets. Functional analysis of key cancer genes exhibiting copy alterations across the cell lines revealed activation/disruption of ten potentially targetable immuno-oncogenic pathways. Genome guided in silico therapy that putatively target these pathways is elucidated. Based on our analysis, five CNV-genes associated with worst survival prognosis are proposed as potential prognostic markers of DLBCL.

Download Full-text