Prognostic values and clinical implications of programmed cell death-ligand 1 (PD-L1), fork head transcription factor P-1 (FOXP-1) and signal transducer and activator of transcription-3 (STAT-3) expression in diffuse large B-cell lymphoma (DLBCL); an immunohistochemical study

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.

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Mitochondrial-Mediated Apoptosis in Lymphoma by the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographolis Paniculata.

Blood ◽

10.1182/blood.v114.22.2710.2710 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2710-2710

Author(s):

Shuo Yang ◽

Andrew M Evens ◽

Savita Bhalla ◽

Sheila Prachand ◽

Amareshwar Singh ◽

...

Keyword(s):

Cell Death ◽

Conformational Change ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Parp Cleavage ◽

Ros Production ◽

Caspase Activation ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 2710 Poster Board II-686 Introduction/Background: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China and other Asian countries to treat a range of diseases, such as respiratory infection, fever, bacterial dysentery and diarrhea. The major bioactive component extracted from Andrographis paniculata is andrographolide. The three hydroxyls at C-3, C-19 and C-14 are responsible for its biological activities. Recently, the anti-cancer properties of andrographolide have been recognized, and it has been tested in human patients. We hypothesized that the mechanism of cell death may depend on caspase activation and mitochondrial mediated cell death pathways. Methods: Using cells lines Ramos (p53 mutated Burkitts lymphoma (BL)), HF-1 (follicular lymphoma (FL)), SUDHL-4 (diffuse large B-cell lymphoma (DLBCL)) and Granta (mantle cell lymphoma (MCL)) we measured cellular cytotoxicity by MTT and apoptosis and quantified by Annexin V-FITC and PI with flow cytometry using FACS. Reactive oxygen species (ROS) production was determined by oxidation of H2DCFDA to dichlorofluorescein (DCF) and quantified by fluorescence intensity and read by flow cytometry. We investigated the mechanism of apoptosis in lymphoma cell lines and patient samples by measuring caspase activation and PARP cleavage by Western blot and Bax conformational change by immunoflourescence. Further, we dissected the influence of the Bax/Bak pathway by using Bax−/−/Bak−/− mouse endothelial fibroblasts (MEFS). Results: We found that andrographolide inhibited survival in all cell lines and was dose and time dependent (IC50 from 15-40uM in cell lines), and was accompanied by ROS production. Cell death was a result of apoptosis and was also dose and time dependent and inhibited by the anti- oxidant N-acetyl-L-cysteine (NAC) and by the pan-caspase inhibitor Z-VAD-FMK and enhanced by the glutathione (GSH) depleting agent buthionine sulfoximine (BSO) and accompanied by PARP cleavage. Similar data were extant in fresh samples from patients with FL, DLBCL, and MCL and there was activation of caspases 3, 8 and 9 in all cell lines and in all patient samples. In order to determine if mitochondrial pathways are involved in cell death, we studied Bax conformational change with the 6A7 monoclonal antibody, which specifically binds Bax protein with conformational change. We found that andrographolide induced Bax conformational change in SUDHL4 and HF1 and similarly in diffuse large B-cell lymphoma and follicular lymphoma pt samples and that this was accompanied by translocation of Bax to the mitochondria in SUDHL-4 (Figure bottom left) and HF-1 (Figure bottom right). Next, using mouse endothelial fibroblast (MEFs) Bax/Bak knockouts (MEFBAX−/−/BAK−/−), we found that andrographolide-induced apoptosis (Figure top left) and change in mitochondrial membrane potential (Figure top right) was mediated through Bax/Bak. Conclusion: This is the first demonstration that andrographolide causes ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, and the mechanism appears to proceed through intrinsic and extrinsic caspase pathways and is associated with Bax/Bak mitochondrial translocation. Further studies of diterpenoid lactones in lymphoma are warranted. Disclosures: Gordon: Cure Tech: Membership on an entity's Board of Directors or advisory committees.

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MiR-181a Is a Master Regulator of the Nuclear Factor-κB Signaling Pathway in Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.417.417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 417-417

Author(s):

Goldi A. Kozloski ◽

Xiaoyu Jiang ◽

Karen L. Bunting ◽

Ari M. Melnick ◽

Izidore S Lossos

Keyword(s):

Cell Death ◽

Cell Lines ◽

Binding Sites ◽

Luciferase Activity ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Reporter Activity ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 417 MicroRNAs (miRNAs) exhibit differential expression in cancer and can be used as prognostic biomarkers. MiR-181a expression is reported to be associated with survival and outcome in acute myeloid and chronic lymphocytic leukemia patients. We demonstrated that miR-181a levels are independently associated with improved survival of diffuse large B cell lymphoma (DLBCL) patients treated with R-CHOP (Rituximab, Cyclophosphamide, Adriamycin, Oncovin, Prednisolone). However, the mechanism underlying this observation and the function of miR-181a in DLBCL pathogenesis are unknown. MiR-181a was expressed at higher levels in centroblasts compared to naïve and memory B cells, and at significantly higher levels in GCB-like compared to ABC-like DLBCL cell lines (p=0.017). These observations suggested that miR-181a may differentially target critical signaling pathways in GCB and ABC DLBCL. NF-kB serves a critical role in ABC DLBCL survival. Utilizing 3 miRNA target prediction algorithms, multiple NF-κB signaling pathway transcripts harbored putative miR-181a binding sites. Consequently, we tested the effect of miR-181a on CARD11, IBKα, p105/p50, and C-Rel expression in DLBCL cell lines (HBL1, VAL). Compared with a scrambled miRNA control, miR-181a expression decreased protein and mRNA levels of these targets. To confirm the effect was direct, we fused the 3′-UTR sequences of CARD11, IBKα, p105 and C-Rel, each containing miR-181a putative binding sites, to a luciferase reporter gene. Co-transfecting miR-181a with the corresponding constructs, we demonstrated that all the constructs had significantly repressed luciferase activity compared with a non-targeting control. The effect was specific, since miR-181a did not affect luciferase activity of CARD11, IBKα, p105 and C-Rel reporter constructs with mutated binding sites. Using an NF-κB luciferase reporter assay, we next demonstrated that compared to a scrambled control, miR-181a significantly decreased NF-κB reporter activity in DLBCL cell lines (VAL, SUDHL6, OCILY7, OCILY19, HBL1, RCK8). MiR-181a also decreased NF-κB reporter activity induced by anti-IgM and TNFα stimulation. Concordantly, anti-miR-181a increased endogenous p105/p50 and C-Rel protein levels. Because ubiquitinated-IKKγ drives NF-κB signaling, we tested the effect of miR-181a in TNFα-stimulated 293T cells on ubiquitinated-IKKγ. MiR-181a decreased levels of ubiquitinated-IKKγ, corroborating the observed inhibitory effects on NF-κB signaling. We reasoned that NF-κB signaling repression should coincide with a decrease in endogenous transcription activity from NF-κB promoters. Indeed, miR-181a decreased mRNA expression levels of NF-κB target genes (BCL2, IRF4, IL-6, IKBa, FN1, PIM1, BLR1, CCL3, CFLAR, FCER2, TP53) as measured by qRT-PCR in miR-181a-transfected HBL1 cells. Because miR-181a directly targets p105/p50 and REL proteins, we postulated that this may be one of the main mechanisms of NF-κB signaling repression. Indeed, an electrophoresis mobility shift assay along with super-shifts analyses showed a decrease in the p105/p50 protein in HeLa nuclear extracts. To examine the biological significance of differential miR-181a expression between GCB- and ABC-like DLBCL and elucidate its potential role in DLBCL pathogenesis, we next assessed cell death (Annexin V, 7AAD) and cell proliferation (BrdU, 7AAD) in GCB (SUDHL4, OCILY7, OCILY19, VAL) and ABC (HBL1, OCILY10, RCK8, U2932) DLBCL cell lines transfected with GFP labeled precursor miR-181a. MiR-181a expression significantly increased cell death and apoptosis of ABC versus GCB DLBCL (p=0.006). This was associated with a more pronounced G1 phase growth arrest in the ABC DLBCL cells. Our studies demonstrate that miR-181a is a master regulator of canonical NF-kB signaling by regulating the expression of multiple components of this pathway, an effect that may underlie the distinct prognosis of DLBCL with different miR-181a expression levels. Furthermore, miR-181a down regulation may contribute to the pathogenesis of ABC DLBCL. Disclosures: No relevant conflicts of interest to declare.

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