Minit-Livin Conjugated to Targeted Nanoparticles Inhibits Tumorigenicity

Abstract We found that Livin, an Inhibitor of Apoptosis Protein, is specifically cleaved to produce a truncated Livin protein (tLivin) and demonstrated the paradoxical pro-apoptotic activity of tLivin. mini-tLivin (mtLivin) is a 70 aa derivative of tLivin that remarkably, exhibits a pro-apoptotic activity as potent as tLivin. Our findings regarding tLivin and mtLivin suggest unique therapeutic avenue against cancer. To improve the delivery and efficacy of mtLivin for treatment we conjugated mtLivin-to nanoparticles (NPs) of poly (lactide-co-glycolide) (PLGA) and targeted the nano-delivery system to diffuse large B-cell lymphoma (DLBCL) using CD40L. Delivery of mtLivin with nanoparticles should enhance stability, provide prolonged bioavailability, intracellular uptake and efficacy. We generated bifunctional mtLivin-CD40L-NPs to target mtLivin-NPs to DLBCL cells. Various cell lines were used to evaluate the biological activity of free and conjugated (targeted or not) mtLivin including 293T, 721.221 B cells, L428 Hodgkin's lymphoma, OCI-LY19 DLBCL cells and OCI-LY19- GLuc cells that constitutively express a gaussia luciferase reporter. Targeted, bifunctional mtLivin-CD40L-NPs elicited significant cell death of DLBCL cells while monofunctional mtLivin-NPs had a lower effect. mtLivin-NPs may induce cell death of some cell lines without targeting (e.g. 293T) yet targeting is required to induce marked cell death of DLBCL cells. To best mimic lymphoma disease in humans, a disseminated lymphoma model was used to evaluate the antitumor effect of targeted mtLivin treatment. NOD/SCID mice were injected with OCI-LY19- GLuc cells or into the tail vein. Gaussia luciferase reporter allows IVIS imaging to monitor tumor size and location in mice. Mice were treated approximately once a week with various mtLivin-NPs formulations. Luciferase signal was observed as early as day 2 after tumor cells injection. Immunohistochemistry for anti-CD20, a human B-cell marker, showed infiltration of OCI-ly19-Gluc cells in spleen and brain close to tumor cells injection (Day 2). With time, lymphoma infiltrations were detected in brain, bone marrow, lungs, spleen and liver. All control mice exhibited paralysis and did not survive beyond 28 days. In contrast, 37.5% of animals receiving mtLivin-NPs and 71.4 % of animals receiving mtLivin-CD40L-NPs achieved complete pathological tumor response and survived significantly longer. All treatments in this study were well-tolerated. In terms of mechanism, we show high caspase-3 activity in tumors from mice that were treated with mtLivin-CD40L-NPs demonstrating the ability of the nanoparticles to target the tumors and to induce apoptotic tumor cell death. The resistance of tumor cells to drug-induced apoptosis is a major cause of cancer treatment failure. We designed mtLitvin targeted nanoparticles constituting novel personalized therapeutic approach for resistant cancer. Disclosures No relevant conflicts of interest to declare.

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Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽

10.1182/blood.v110.11.4525.4525 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4525-4525

Author(s):

Bernardo Martinez-Miguel ◽

Melisa A. Martinez-Paniagua ◽

Sara Huerta-Yepez ◽

Rogelio Hernandez-Pando ◽

Cesar R. Gonzalez-Bonilla ◽

...

Keyword(s):

Cell Death ◽

B Cells ◽

Fusion Protein ◽

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

B Cell Lymphoma ◽

Genetically Engineered ◽

B Cell Malignancies ◽

Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

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

Blood ◽

10.1182/blood.v104.11.242.242 ◽

2004 ◽

Vol 104 (11) ◽

pp. 242-242 ◽

Cited By ~ 4

Author(s):

Hovav Nechushtan ◽

Joseph D. Rosenblatt ◽

Izidore S. Lossos

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Lysis ◽

Large B Cell Lymphoma ◽

Expression Of Genes ◽

Large B Cell

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

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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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T Cells Expressing CD19-Specific Chimeric Antigen Receptors Are Inhibited By Indoleamine 2,3-Dioxygenase in Tumors

Blood ◽

10.1182/blood.v124.21.2434.2434 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2434-2434

Author(s):

Soranobu Ninomiya ◽

Leslie E Huye ◽

Barbara Savoldo ◽

Gianpietro Dotti ◽

Helen E. Heslop ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Tumor Control ◽

Intracellular Enzyme ◽

Indoleamine 2,3 Dioxygenase

Abstract Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that mediates the metabolism of tryptophan to kynurenines, which have an inhibitory activity on immune cells. IDO-positive tumors thus establish a microenvironment in which NK and T cells are inactivated, and high IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in diffuse large B-cell lymphoma patients. CD19-specific chimeric antigen receptor T cell (CART) therapy is a promising new approach against B-cell malignancies but not every tumor responds. To determine whether the presence of IDO in tumors affects CART activity, we first investigated the expression of IDO by the human B-cell lymphoma cell lines Raji, Daudi, BJAB and Jeko-1. We found that only Jeko-1 expresses IDO and produces kynurenine natively. IDO was not expressed by the other cell lines, even after exposure to IFNγ, a known IDO inducer. Based on these results, we chose Raji as a baseline IDO-negative cell line and made an IDO-positive Raji clone by retroviral transduction with human IDO cDNA (Raji-IDO); a clone transduced with an empty vector served as control (Raji-control). We injected SCID mice subcutaneously in opposite flanks with luciferase-transduced Raji-control and Raji-IDO cells. Seven days later, we injected human non-transduced T cells (NTs) or CARTs intravenously. In the NT group, tumors had similar growth on both sides. In contrast, in the CART group, Raji-control tumors had significantly slower growth than Raji-IDO tumors (3.1 ± 1.1×108 and 20 ± 7.3×108 bioluminescence units [BU] at day 7, respectively, P = 0.03). We also found that CARTs significantly inhibited Raji-control tumor growth (NT: 27 ± 6.8×108 vs CART: 3.1 ± 1.1×108 BU at day 7, P = 0.02), but did not affect Raji-IDO tumors (NT: 24 ± 5.4×108 vs CART: 20 ± 7.3×108 BU at day 7, P = 0.35). In another experiment, Raji-IDO cells were injected subcutaneously, and mice were treated with an IDO inhibitor (1-methyl-tryptophan, 1-MT), CARTs, or both. The combination treatment produced significantly better tumor control than either single therapy (1-MT: 45 ± 6.8×108 and CART: 22 ± 4.6×108 vs both: 8.2 ± 3×108 BU at day 7, P = 0.001 and 0.04, respectively). Thus, the IDO inhibitor protects CARTs against the deleterious effects of IDO-positive tumors. To investigate potential mechanisms for CART inhibition by IDO, we assessed the effect of kynurenine and found that even low concentrations (12.5 µM) inhibited CART proliferation in response to IL-2, IL-7, IL-15 or CD19-positive targets, although there was no effect on proliferation of B-cell lymphoma cell lines at this kynurenine concentration. CART apoptosis was also increased by kynurenine (8.6 ± 0.6%, 13.9 ± 2.2%, and 33.5 ± 10.6% annexin V-positive cells with 0, 12.5, or 25 µM kynurenine). In coculture of CARTs with wild-type Raji cells, the latter were eliminated by day 6 in the absence of kynurenine, but increased in numbers (in parallel with a decrease in CARTs) in its presence. Kynurenine also inhibited the release of IFNγ (13,143 ± 848 pg/mL vs 2,663 ± 1,873 pg/mL, P = 0.02) and IL-2 (718 ± 355 pg/mL and 199 ± 165 pg/mL, P = 0.03) by CARTs. Expression of granzyme B, PD-1 and CTLA-4 on CARTs was not significantly affected. Fludarabine has been reported to downregulate IDO expression in tumors and this drug is used in many lymphodepleting regimens that are administered before CART infusion in an effort to augment the efficacy of these therapies. However, the beneficial mechanism of lymphodepleting chemotherapy drugs is not fully understood. Therefore, we also measured the effect of fludarabine and mafosfamide (a cyclophosphamide analog) on IDO expression by Jeko-1 cells. We found that both drugs downregulate IDO expression by Jeko-1, even in the presence of IFNγ. In summary, expression of IDO by tumor cells inhibits CART activity, likely because kynurenine is produced and has negative effects on proliferation and cytokine secretion by CARTs. Fludarabine and cyclophosphamide downregulate IDO expression in tumors and this effect may contribute to the benefits of lymphodepletion before CART therapy. Direct IDO inhibitors may further improve clinical CART activity against IDO-positive tumors. Disclosures No relevant conflicts of interest to declare.

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Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Blood ◽

10.1182/blood.v124.21.2963.2963 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2963-2963

Author(s):

Goldi A Kozloski ◽

Xiaoyu Jiang ◽

Shruti Bhatt ◽

Rita Shaknovich ◽

Ari M Melnick ◽

...

Keyword(s):

B Cell ◽

Signaling Pathway ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Large B Cell Lymphoma ◽

Animal Survival ◽

Dlbcl Subtype ◽

Large B Cell

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis. Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. Figure 1 Figure 1. Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.3022.3022 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3022-3022

Author(s):

Jonathan Scott Rink ◽

Sol Misener ◽

Osman Cen ◽

Shuo Yang ◽

Leo I. Gordon ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Density Lipoprotein ◽

B Cell Lymphoma ◽

Cell Receptor ◽

Cholesterol Homeostasis ◽

B Cell Lymphomas ◽

Bcr Signaling

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

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Lw-213 Synergizes with Rituximab to Inhibit Diffuse Large B-Cell Lymphomas By Upregulating CD20

Blood ◽

10.1182/blood-2021-149515 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4366-4366

Author(s):

Yuchen Li ◽

Hui Li ◽

Hui Hui ◽

Jingyan Xu

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

B Cell Lymphoma ◽

Cell Counting ◽

Mrna Levels ◽

Sox2 Expression ◽

Cd20 Expression ◽

Large B Cell

Abstract Background：Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens.Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, since the remaining therapeutic options are limited. LW-213, a derivative of wogonin, is reported to possess antineoplastic properties in a variety of cancers, but whether it has effects on DLBCL is n-ot known. Studies have reported that upregulation of CD20 expression b-y either HDACi or silenced SOX2 expression showed sensitizing potential in Rituximab-induced cell death in malignant B cells. Our study was to explore whether LW-213 could sensitize DLBCL to Rixutimab thus improve therapeutic efficacy. Methods： Two DLBCL cell lines, RI-1 (ABC subtype) and Su-DHL -8 (GCB subtype), were used in our study. RI-1 and Su-DHL-8 cells were treated with LW-213 at different doses and for different times, and their proliferation and viability were detected by Cell counting kit-8 (CCK8).Flow cytometry was used to determine surface CD20 expression. Western blotting and q-PCR were applied to examine the protein and mRNA levels of CD20, SOX2, Ace-H3 and Ace-H3K27. CDC assay was used to evaluate the synergistic effects of LW-213 and Rixutimab. Results：We showed that LW-213 inhibited the proliferation of human DLBCL cell lines (Su-DHL-8、RI-1 ) in dose-and time-dependent manners with IC 50 values at the low μmol/L levels, meanwhile it potently inhibited primary lymphoma cells derived from peripheral blood of B-cel-l lymphoma patients. Furthermore, LW-213 significantly increased CD20 surface expression and the acetylation level of histone in DLBCL cell li-nes. Inversely，the SOX2 expression level remarkably decreased. Finally,Combination with LW-213 significantly synergized Rituximab-induced cell death in vitro. Conclusion： The results demonstrate that LW-213 sensitizes DLBCL cells to Rituximab in vitro by upregulating CD20 expression and the SOX2/ace-H3K27/ace-H3 axis may plays a critical role in CD20 upregulation processing. Even though this strategy is important in vitro models,the upregulating CD20 expression therapy against DLBCL proposed in this study warrants further study in vivo and clinical trials . Keywords：CD20 DLBCL Rituximab SOX2 Histone Deacetylation Disclosures No relevant conflicts of interest to declare.

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Circular RNA hsa_circ_0000282 contributes to osteosarcoma cell proliferation by regulating miR-192/XIAP axis

BMC Cancer ◽

10.1186/s12885-020-07515-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Houkun Li ◽

Limin He ◽

Yuan Tuo ◽

Yansheng Huang ◽

Bing Qian

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Circular Rnas ◽

Control Group ◽

Osteosarcoma Cell ◽

Luciferase Reporter Gene ◽

Apoptosis Protein ◽

Gene Assay

Abstract Background Circular RNAs (circRNAs) have emerged as a novel category of non-coding RNA, which exhibit a pivotal effect on regulating gene expression and biological functions, yet how circRNAs function in osteosarcoma (OSA) still demands further investigation. This study aimed at probing into the function of hsa_circ_0000282 in OSA. Methods The expressions of circ_0000282 and miR-192 in OSA tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the correlation between the expression level of circ_0000282 and clinicopathological features of OSA patients was analyzed. The expressions of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in OSA cells were assayed by Western blot. The proliferation and apoptosis of OSA cells were examined by CCK-8, BrdU and flow cytometry, respectively. Bioinformatics analysis, dual-luciferase reporter gene assay and RIP experiments were employed to predict and validate the targeting relationships between circ_0000282 and miR-192, and between miR-192 and XIAP, respectively. Results Circ_0000282 was highly expressed in OSA tissues and cell lines, which represented positive correlation with Enneking stage of OSA patients and negative correlation with tumor differentiation degree. In vitro experiments confirmed that overexpression of circ_0000282 markedly facilitated OSA cell proliferation and repressed cancer cell apoptosis in comparison to control group. Besides, knockdown of circ_0000282 repressed OSA cell proliferation and promoted apoptosis. Additionally, the binding relationships between circ_0000282 and miR-192, and between miR-192 and XIAP were validated. Circ_0000282 indirectly up-regulated XIAP expression by adsorbing miR-192, thereby playing a role in promoting cancer in OSA. Conclusion Circ_0000282 was a novel oncogenic circRNA in OSA. Circ_0000282/miR-192/XIAP axis regulated OSA cell proliferation apoptosis with competitive endogenous RNA mechanism.

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Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphomas

Modern Pathology ◽

10.1038/s41379-020-0575-3 ◽

2020 ◽

Vol 33 (12) ◽

pp. 2407-2421 ◽

Cited By ~ 1

Author(s):

Lucia Mundo ◽

Leonardo Del Porro ◽

Massimo Granai ◽

Maria Chiara Siciliano ◽

Virginia Mancini ◽

...

Keyword(s):

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

High Sensitivity ◽

B Cell Lymphoma ◽

Rare Tumor ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr ◽

Higher Sensitivity

AbstractThe Epstein–Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a “hit-and-run” mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged.

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