BJAB Cells Research Articles

CXCR4 Antagonist BKT140 Synergizes with Rituximab, Targeting Non Hodgkin Lymphoma (NHL) In Vitro and In Vivo in a Xenograft Model with Bone Marrow Involvement

Abstract 952 Background:B-cell Non Hodgkin lymphoma (NHL) represents the most common malignant lymphoid neoplasm, with heterogeneous pathophysiological and clinical presentation. It may show systemic, nodal or extra nodal localization in various sites, including bone marrow (BM). Although anti-CD20 antibody rituximab significantly improved the outcome of NHL patients, the relapsed/refractory rates are still high. Chemokine receptor CXCR4 and its ligand CXCL12 are critically involved in the survival and trafficking of normal and malignant B lymphocytes. Taking together with the fact that interaction of malignant B cells with stromal cells via CXCR4/CXCL12 signaling may provide chemo-resistance, we hypothesized that blockade of CXCR4 may antagonize the survival and spreading of lymphoma cells and restore their chemo-sensitivity. Results:The effect of CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity was tested in vitro and in vivo. Inhibition of CXCR4 with BKT140 in CD20-expressing lymphoma cell lines (BL-2, BJAB, Raji, Ramos, SUDHL4, OCI-Ly7) and primary lymphoma cells from patients with BM involvement resulted in significant inhibition of cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the cytotoxic effect (apoptosis) against the lymphoma cells in a dose-dependent manner (p<0.01). This effect was concurrent with increase in Annexin V binding, loss of ΔΨm and caspase 3 cleavage. These findings indicate that mitochondrial membrane dysfunction and caspase 3 activation are involved in BKT140- and rituximab-mediated lymphoma cell death. Moreover, we were able to show that rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, both on mRNA and cell-surface levels, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. This interaction provides the rationale for CXCR4 targeting in combination with rituximab treatment. Indeed, highly-expressing CXCR4 BL-2 cells were protected by BM stromal cells from rituximab-induced apoptosis, whereas low-CXCR4 BJAB cells were not. The protective effect of stoma was abrogated by BKT140. To evaluate the in vivo anti-lymphoma effect of BKT140 we established a xenograft model of B-cell lymphoma with BM involvement in mice. Human CXCR4-expressing B NHL cell line, BL-2, was subcutaneously implanted into NOD/SCID mice, resulting in the development of aggressive local tumors which specifically spread to the bone marrow. Following the tumor establishment, mice were injected subcutaneously with BKT140, rituximab or with combination of both. BKT140 inhibited the local tumor progression of BL-2 generated tumors. Furthermore, BKT140 treatment significantly reduced the number of BL-2 tumor cells in the BM by 77% (p<0.01) compared to the untreated mice, while rituximab decreased this number only by 20%. Importantly, the combination treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the BM, achieving 93% reduction (p<0.012). We found that BKT140 promoted lymphoma cell apoptosis within the bone marrow microenvironment. Furthermore, we found that the number of viable lymphoma cells in the peripheral blood of tumor-bearing animals was also reduced following the treatment with BKT140. Conclusions:Our results demonstrate potent anti-lymphoma effect of CXCR4-specific antagonist BKT140 in vitro and in vivo. The BKT140-mediated anti-lymphoma effect synergizes with that of Rituximab. Moreover, BKT140 effectively targets lymphoma cells in the bone marrow microenvironment, overcoming the stroma-induced resistance to rituximab. These findings suggest the possible interaction between CD20 and CXCR4 pathways in NHL, and provide the scientific basis for the development of novel combined CXCR4-targeted therapies for refractory NHL. Disclosures:No relevant conflicts of interest to declare.

The role of the intrinsic FAS pathway in Titanocene Y apoptosis: The mechanism of overcoming multiple drug resistance in malignant leukemia cells

Despite the advantages in the outcome of patients with acute lymphoblastic leukemia, 25% of the affected children suffer relapses. As the response to chemotherapy is essentially determined by the development of cellular drug resistance, new drugs that are capable to overcome resistance to conventional chemotherapeutics are urgently needed. With regard to this demand, we investigated the titanium-based anticancer drug Titanocene Y.Treatment with Titanocene Y leads to inhibition of tumour cell proliferation and induces apoptosis in established cell lines of leukemia, lymphoma and melanoma.The extrinsic pathway appears to be responsible, at least in part, for the effect: cell death is partly inhibited in BJAB cells overexpressing a dominant negative Fas-associated death domain (FADD) mutant and via real time PCR we found a significant up-regulation of Fas ligand in the affected cells. Apoptosis is triggered regardless of the expression of anti-apoptotic Bcl-2 and pro-apoptotic smac and the agent is also effective on cells that are multidrug resistant due to overexpression of P-gp. In combination with vincristine impressive synergistic effects could be observed, exposing Titanocene Y as a possible component for polychemotherapy.Taken together, Titanocene Y turns out to be a promising candidate for anti-tumour therapy, especially for the treatment of multidrug resistant malignancies.

Human periodontal ligament fibroblasts stimulate osteoclastogenesis in response to compression force through TNF-α-mediated activation of CD4+ T cells

Periodontal ligament fibroblasts (PLF) sense and respond to mechanical stimuli and participate in alveolar bone resorption during orthodontic treatments. This study examined how PLF influence osteoclastogenesis from bone marrow-derived macrophages (BMM) after application of tension or compression force. We also investigated whether lymphocytes could be a primary stimulator of osteoclastic activation during alveolar bone remodeling. We found that mechanical forces inhibited osteoclastic differentiation from BMM in co-cultures with PLF, with PLF producing predominantly osteoprotegerin (OPG) rather than receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). In particular, PLF increased the expression of tumor necrosis factor (TNF)-α in response to compression. Additional experiments showed the presence of CD4- and B220-positive cells with a subsequent increase in tartrate-resistant acid phosphatase (TRAP)-positive cells and RANKL expression only at the compression side of the force-subjected periodontal tissues. Exogenous TNF-α increased the number of TRAP-positive cells and pit formation in the co-cultures of BMM with Jurkat, but not with BJAB cells and this effect was almost completely inhibited by antibodies to TNF-α or TNF receptor. Collectively, the current findings suggest that PLF secrete relatively higher levels of TNF-α at the compression side than at the tension side and this imbalance leads to RANKL expression by activating CD4+ T cells, thereby facilitating bone resorption during orthodontic tooth movement.

Infection of Lymphoblastoid Cell Lines by Kaposi's Sarcoma-Associated Herpesvirus: Critical Role of Cell-Associated Virus

Kaposi's sarcoma-associated herpesvirus (KSHV) displays strong lymphotropism in vivo, but paradoxically, established B cell lines have largely been refractory to infection by soluble KSHV virions. Here we show that this block can be overcome by exposure to cell-associated virus. Doxycycline-inducible recombinant KSHV.219 (rKSHV.219)-harboring SLK (iSLK.219) cells were employed as KSHV donors. Cocultivation of lymphoid cell lines with reactivated iSLK.219 cells resulted in readily demonstrable viral entry into each cell line; similar observations were made in primary tonsillar B cell cultures. Moreover, infected lymphoid cells were able to outgrow upon puromycin selection, indicating development of persistent infection. Infected BJAB cells display signatures of latent infection, including classical latency-associated transcripts, a punctate pattern of LANA expression, and episomal maintenance of the KSHV genome. However, when lytically activated by various chemical stimuli, infected BJAB cells were able to produce only low levels of infectious virions. These data demonstrate that (i) cell-associated viruses can bypass viral entry blocks in most lymphoid cell lines, (ii) the determinants of cell-associated virus entry differ from those of soluble virion infection, and (iii) immortalized lymphoblastoid lines have partial postentry blocks to efficient lytic reactivation.

Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

BackgroundCucurbitacin-I (JSI-124) is potent inhibitor of JAK/STAT3 signaling pathway and has anti-tumor activity in a variety of cancer including B cell leukemia. However, other molecular targets of JSI-124 beyond the JAK/STAT3 pathway are not fully understood.MethodsBJAB, I-83, NALM-6 and primary CLL cells were treated with JSI-124 as indicated. Apoptosis was measured using flow cytometry for accumulation of sub-G1 phase cells (indicator of apoptosis) and Annexin V/PI staining. Cell cycle was analyzed by FACS for DNA content of G1 and G2 phases. Changes in phosphorylation and protein expression of p38, Erk1/2, JNK, c-Jun, and XIAP were detected by Western blot analysis. STAT3 and c-Jun genes were knocked out using siRNA transfection. VEGF expression was determined by mRNA and protein levels by RT-PCR and western blotting. Streptavidin Pull-Down Assay was used to determine c-Jun binding to the AP-1 DNA binding site.ResultsHerein, we show that JSI-124 activates c-Jun N-terminal kinase (JNK) and increases both the expression and serine phosphorylation of c-Jun protein in the B leukemic cell lines BJAB, I-83 and NALM-6. JSI-124 also activated MAPK p38 and MAPK Erk1/2 albeit at lower levels than JNK activation. Inhibition of the JNK signaling pathway failed to effect cell cycle arrest or apoptosis induced by JSI-124 but repressed JSI-124 induced c-Jun expression in these leukemia cells. The JNK pathway activation c-Jun leads to transcriptional activation of many genes. Treatment of BJAB, I-83, and NALM-6 cells with JSI-124 lead to an increase of Vascular Endothelial Growth Factor (VEGF) at both the mRNA and protein level. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Pretreatment with recombinant VEGF reduced JSI-124 induced apoptosis.ConclusionsTaken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to increased VEGF expression.

Functional FCGR2B gene variants influence intravenous immunoglobulin response in patients with Kawasaki disease

In Kawasaki Disease patients, the authors show associations between high-dose intravenous immunoglobulin (IVIG) response and a polymorphism in the FCγRIIB. This provides basis for defining the IVIG regulatory mechanisms and pharmacogenomic approach to IVIG therapy.

EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding

EBV nuclear antigen 2 (EBNA2) and EBV nuclear antigen LP (EBNALP) are critical for B-lymphocyte transformation to lymphoblastoid cell lines (LCLs). EBNA2 activates transcription through recombination signal-binding immunoglobulin κJ region (RBPJ), a transcription factor associated with NCoR repressive complexes, and EBNALP is implicated in repressor relocalization. EBNALP coactivation with EBNA2 was found to dominate over NCoR repression. EBNALP associated with NCoR and dismissed NCoR, NCoR and RBPJ, or NCoR, RBPJ, and EBNA2 from matrix-associated deacetylase (MAD) bodies. In non-EBV-infected BJAB B lymphoma cells that stably express EBNA2, EBNALP, or EBNA2 and EBNALP, EBNALP was associated with hairy and enhancer of split 1 (hes1), cd21, cd23, and arginine and glutamate-rich 1 (arglu1) enhancer or promoter DNA and was associated minimally with coding DNA. With the exception of RBPJ at the arglu1 enhancer, NCoR and RBPJ were significantly decreased at enhancer and promoter sites in EBNALP or EBNA2 and EBNALP BJAB cells. EBNA2 DNA association was unaffected by EBNALP, and EBNALP was unaffected by EBNA2. EBNA2 markedly increased RBPJ at enhancer sites without increasing NCoR. EBNALP further increased hes1 and arglu1 RNA levels with EBNA2 but did not further increase cd21 or cd23 RNA levels. EBNALP in which the 45 C-terminal residues critical for transformation and transcriptional activation were deleted associated with NCoR but was deficient in dismissing NCoR from MAD bodies and from enhancer and promoter sites. These data strongly support a model in which EBNA2 association with NCoR-deficient RBPJ enhances transcription and EBNALP dismisses NCoR and RBPJ repressive complexes from enhancers to coactivate hes1 and arglu1 but not cd21 or cd23.

Abstract 2651: Cucurbitacin I (JSI-124) activates the JNK signaling pathway leading to transcriptional up-regulation of vascular endothelial growth factor in B leukemia cells

Abstract JSI-124 (Cucurbitain I) is a biochemical compound isolated from the herbal plant families, which have been used as folk medicines for centuries in Asian countries. The molecular mechanisms for this inhibitor acts primarily through STAT3 inhibition in many tumors, including chronic lymphocytic leukemia. However, other molecular targets of JSI-124 are not fully understood. The objective of this study was to determine the molecular targets of JSI-124 beyond STAT3. Chemotherapeutic drugs often induce a stress response in cancer cells. One of early stress response pathways is the c-Jun N-terminal kinase (JNK) pathway. Herein, we show that JSI-124 activates JNK and increased the expression of c-Jun protein in the B leukemia cell lines BJAB, I-83 and NALM-6. Interestingly, the inhibition of the JNK signaling pathway failed to effect JSI-124 induced cell cycle arrest or apoptosis in these leukemia cells but repressed c-Jun level. Furthermore, JNK activation also failed to effect JSI-124 inhibition of STAT3 phosphorylation in these cells. It is known that one of the important factors involved in JNK signaling is Vascular Endothelial Growth Factor (VEGF). We found that JSI-124 treated BJAB cells showed an increased in mRNA and protein level in VEGF in BJAB, I-83 and NALM-6 cells. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Taken together, our data demonstrate that JSI-124 activates JNK signaling pathway leading to VEGF expression independent of inhibition of STAT3 activation. This could lead to increased angiogenesis in B leukemia cells treated with JSI-124. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2011-2651

Catechin-7-O-β-d-glucopyranoside scavenges free radicals and protects human B lymphoma BJAB cells on H2O2-mediated oxidative stress

Catechin-7-O-β-d-glucopyranoside (CA-G) was previously isolated from red bean (the seed of Phaseolus calcaratus cv. Roxburgh). This study examined the ability of CA-G to scavenge reactive oxygen species generated by cell-free systems and to protect cells from oxidative stress caused by hydrogen peroxide (H2O2). The mechanism by which CA-G exerts its antioxidant and anti-apoptotic action on H2O2-exposed cells was also investigated. CA-G treatment prevented H2O2-mediated apoptosis and inhibited the formation of single stand breaks in DNA in H2O2-exposed BJAB cells. CA-G suppressed mitochondrial stress and caspase activation caused by H2O2. Mechanistic experiments revealed that the antioxidant mechanism of CA-G on H2O2-mediated oxidative damage was due to the direct scavenging of hydroxyl radicals and/or to the chelation of metal ions that were used to produce hydroxyl radicals from H2O2 via the Fenton reaction. Collectively, these findings suggest beneficial roles of CA-G or CA-G-rich red bean on the protection from oxidative damage.

NF-κB Down-regulates Expression of the B-lymphoma Marker CD10 through a miR-155/PU.1 Pathway

Cell-surface protein CD10 is a prognostic marker for diffuse large B-cell lymphoma (DLBCL), where high expression of CD10 is found in the germinal center B-cell (GCB) subtype and CD10 expression is low or absent in the activated B-cell (ABC) subtype. As compared with the GCB subtype, patients with ABC DLBCL have a poorer prognosis after standard treatment, and ABC tumor cells have higher NF-κB activity. Herein, we show that increased expression of the NF-κB target micro-RNA miR-155 is correlated with reduced expression of transcription factor PU.1 and CD10 in several B-lymphoma cell lines. Moreover, electromobility shift assays and luciferase reporter assays indicate that PU.1 can directly activate expression from the CD10 promoter. Expression of a DLBCL-derived mutant of the adaptor CARD11 (a constitutive activator of NF-κB) in the GCB-like human BJAB cell line or v-Rel in the chicken DT40 B-lymphoma cell line causes reduced expression of PU.1. The CARD11 mutant also causes a decrease in CD10 levels in BJAB cells. Similarly, overexpression of miR-155, which is known to down-regulate PU.1, leads to reduced expression of CD10 in BJAB cells. Finally, we show that CD10 expression is reduced in BJAB cells after treatment with the NF-κB inducer lipopolysaccharide (LPS). Additionally, miR-155 is induced by LPS treatment or expression of the CARD11 mutant in BJAB cells. These results point to an NF-κB-dependent mechanism for down-regulation of CD10 in B-cell lymphoma: namely, that increased NF-κB activity leads to increased miR-155, which results in decreased PU.1, and consequently reduced CD10 mRNA and protein.

The rare-earth yttrium complex [YR(mtbmp)(thf)] triggers apoptosis via the extrinsic pathway and overcomes multiple drug resistance in leukemic cells

A major problem in treating patients with acute lymphoblastic leukemia is the development of drug resistance. In the current study, we investigated the anticancer properties of the novel rare-earth yttrium complex [YR(mtbmp)(thf)] in various established cell lines, and moreover, we identified the involved apoptotic pathway. Further aim was to investigate whether synergistic effects could be reached in combination with the conventional drug vincristine. We used the yttrium complex [YR(mtbmp)(thf)] in cells of leukemia (Nalm-6) and lymphoma (BJAB) and identified the main mechanism of the apoptosis induction by measuring the amount of hypodiploid DNA via FACS Scan analysis. Exposure of BJAB cells to [YR(mtbmp)(thf)] led to a death receptor-mediated reduction of cell viability and induction of apoptosis. The independence of Bcl-2 expression supports the suggestion that the [YR(mtbmp)(thf)]-induced apoptosis is mainly mediated via the extrinsic pathway. The extensive anti-tumor activity of [YR(mtbmp)(thf)] could be underlined by its capability to overcome multiple drug resistance in leukemic cells (Nalm-6) that are characterized by an overexpression of P-glycoprotein. [YR(mtbmp)(thf)] in combination with the conventional drug vincristine displayed impressive synergistic effects. We demonstrate in vitro efficiency of [YR(mtbmp)(thf)] in cells of hematological malignancies and reveal its ability to be a possible agent for polychemotherapy.

Functional characterization of Kaposi's sarcoma-associated herpesvirus open reading frame K8 by bacterial artificial chromosome-based mutagenesis.

The open reading frame K8 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a basic leucine zipper (bZip) protein that binds to the origin of viral DNA replication and is an integral component of viral lytic DNA replication complex. Moreover, K8 physically interacts with replication and transcription activator (RTA) and represses its transactivation activity on several viral promoters. To investigate the role of this protein in viral life cycle, we constructed two K8-null recombinant mutant viruses (BAC-ΔK8 and BAC-stopK8) by using a bacterial artificial chromosome (BAC) system. Latent viral infection can be reconstituted in 293T and BJAB cells with wild-type and the K8-null recombinant viruses by introducing the cloned viral genomes into the cells. When the cells carrying these viruses were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, no significant difference was seen in overall viral gene expression between wild-type and K8-null viruses, with lytic DNA replication still active in the latter. However, 293T cells harboring K8-null mutant viruses, either BAC-ΔK8 or BAC-stopK8, displayed lower copy numbers of latent KSHV genome in comparison with wild-type viruses. Furthermore, although K8 deficiency appeared to not affect infectivity when K8-null viruses were used to infect 293T, primary human microvascular dermal endothelial and human foreskin fibroblast cells, they exhibited much lower viral genome copy numbers in all types of cell compared to wild-type viruses. Taken together, these data suggest a possible role of K8 in abortive lytic DNA replication occurring in early stages of de novo infection or in the maintenance of latent viral genomes.

Synthesis and DNA-binding properties of apoptosis-inducing cytotoxic half-sandwich rhodium(III) complexes with methyl-substituted polypyridyl ligands

Half-sandwich organorhodium(III) complexes of the type [(η 5-C 5Me 5)RhCl(pp)] (CF 3SO 3) containing polypyridyl ligands (pp) represent a promising class of cytostatic agents. Replacement of the polypyridyl ligands of complexes 1 (pp = phen) and 6 (pp = dppz) by methyl-substituted derivatives in 2– 5 (pp = 4-Mephen, 5-Mephen, 4,7-Me 2phen, 5,6-Me 2phen) and 7 (pp = Me 2dppz) leads to a significant improvement in their antiproliferative activity towards human MCF-7 and HT-29 cancer cells. For instance, the IC 50 value towards HT-29 cells decreases from 4.3 ± 0.2 μM for 6 to 0.98 ± 0.49 μM for complex 7. In contrast, no activity (IC 50 > 100 μM) was observed for the HOOC and n-BuNHCO substituted dppz complexes 8 and 9. UV/vis, CD and NMR spectra for mixtures of complexes 7– 9 with CT DNA were in accordance with intercalation of the substituted dppz ligands between the base pairs of the double helix and direct evidence for this binding mode was also provided by a 2D NOESY study for complex 7 with the hexanucleotide d(5′-CGTCGG-3′). Each of the methyl-substituted phen complexes 2– 5 is significantly more active towards immortalized HEK-293 cells (IC 50 values 0.40 ± 0.02 to 0.94 ± 0.02 μM) than towards the cancer cells. Flow cytometric measurements of DNA fragmentation in BJAB cells following an incubation period of 72 h with 1, 5 and 6 indicate that the complexes induce specific apoptotic cell death in the non-adherent lymphoma cells.

Effect of Valproic Acid (VPA) on Fludarabine Activity In Chronic Lymphocytic Leukemia (CLL) Patients

AbstractAbstract 4626Valproic acid (VPA) is a first-generation anti-epileptic drug that has been shown to inhibit proliferation and induce apoptosis in various hematological malignancies, including multiple myeloma and chronic lymphocytic leukemia (CLL). VPA-induced apoptosis is thought to be due to its ability to inhibit histone deacetylases (HDACs), resulting in hyper-acetylation of histones and altered expression levels of pro- and anti-apoptotic genes. We show here that the mechanism of VPA-induced apoptosis in CLL is associated with activation of the TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway in vitro and ex vivo. TRAIL induces cell death by binding to death receptors 4 and 5 (DR4, DR5), which in turn activate the extrinsic apoptotic pathway. We have previously shown that 30–40% of the activities of fludarabine and chlorambucil are mediated through the TRAIL pathway, and this is related to up-regulation of the expressions of DR4 and DR5. We confirm here that VPA induces apoptosis in two CLL-like cell lines, I-83 and BJAB. Co-treatment of the two cell lines with VPA and activating antibodies against either DR4 (ETR1) or DR5 (ETR2) enhanced VPA-induced apoptosis in both cell lines after 24 hours. Conversely, blocking the TRAIL apoptotic pathway with DR4:FC fusion protein decreased the cytotoxicity of VPA, as well as another HDAC inhibitor, trichostatin A, in BJAB cells. These results confirmed the importance of activation of the TRAIL apoptotic pathway for the antitumor activity of VPA. Treatment with VPA also induced formation of reactive oxygen species (ROS) and enhanced fludarabine-induced apoptosis in cell lines and primary CLL cells. Having shown the effects of VPA in vitro, a phase II clinical trial was initiated at CancerCare Manitoba to determine the activity of VPA in CLL, either when used alone or in combination with fludarabine. Five patients who had received at least one prior therapy with fludarabine have to date been examined. Three out of 5 patients were fludarabine-resistant, as defined as no response to fludarabine or relapse < 6-months after completion of treatment with fludarabine. Patients received VPA at a starting dose of 15 mg/kg/day orally in divided doses, with the goal of reaching a serum level of >1 mM. Various side effects were observed with higher doses of VPA, including anaemia, thrombocytopaenia, GI toxicity and fatigue. No responses were seen after 28 days using VPA alone. However, in four patients who continued on VPA with fludarabine, 3 patients showed a >50% fall in lymphocyte/lymph node size after receiving 5 cycles of the combination. Peripheral blood samples were obtained prior to each treatment cycle, and mononuclear cells were isolated for analysis by immunoblotting and immunocytochemistry. Levels of both histone 3-acetyl and histone 4-acetyl initially increased and then fluctuated during the course of treatment. When DR4 and DR5 levels were examined by immunoblotting, DR4 levels increased during the course of therapy, while no significant changes in DR5 levels were observed. Expression of mRNA levels of genes involved in apoptosis was examined in one patient before and 28 days following VPA. There was a global up-regulation of both pro- and anti-apoptotic genes (as categorized by their gene ontology) which likely explains the effect of VPA on fludarabine sensitivity in CLL. In summary, VPA induces hyper-acetylation of histones in CLL and activates the TRAIL apoptotic pathway through upregulation of DR4 levels. While VPA is ineffective as a single agent in CLL, it can sensitize CLL cells to fludarabine and could be used as an adjuvant in fludarabine-based treatment regimens. Ongoing studies are evaluating the effectiveness of lower and less toxic doses of VPA in combination with fludarabine in CLL. Disclosures:Off Label Use: Valproic acid (VPA). VPA is being tested as a possible adjunct in treating chronic lymphocytic leukemia.

Stroma-Induced Increase In ABCG2 Expression Is Involved In Resistance to Chemotherapy In Diffuse Large B-Cell Lymphoma

AbstractAbstract 972Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of anti-apoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. However, the cellular mechanisms responsible for drug resistance and treatment failure in DLBCL are poorly understood and their elucidation could lead to development of new therapeutic approaches. Activated Hedgehog (HH) signaling was identified in our laboratory to contribute to DLBCL cell survival and proliferation. We also found that inhibiting HH signaling resulted in decrease of BCL-2 protein and mRNA levels in ABC DLBCL cells but not in GC DLBCL cells, and that the resistance to apoptosis to HH inhibition observed in GC DLBCL cells can be overcome using the BCL2 inhibitor YC137. (Leukemia 2010;24:1025). We also found that the drug transporter protein ABCG2 is a potential prognostic factor in DLBCL given that its expression (as detected by immunohistochemistry) inversely correlated with overall survival and failure-free survival (Mod Pathol 2009;22:1312). Here, we investigate potential mechanistic connections between HH signaling and chemoresistance in DLBCL. Using chromatin immunoprecipitation (ChIP), site directed mutagenesis and luciferase reporter based assays, we confirmed the presence of a GLI-1 transcription factor-binding site in the ABCG2 gene promoter and we established ABCG2 gene as a direct downstream target of HH signaling. We also characterized the baseline expression and functionality of ABCG2 in DLBCL cell lines and explored the role of stroma in modulating its expression levels as well as the expression of other drug transporters (MDR1 and ABCC1) and the levels of the anti-apoptotic proteins, BCL-2, BCL2A1 and BCL-xL. Co-culturing DLBCL cell lines (BJAB and DOHH2) with human bone marrow stromal cells (HS5) resulted in decreased chemosensitivity to doxorubicin and methotrexate that was associated to marked increased expression levels of the drug transporters ABCG2, MDR1 and ABCC1 as well as of the expression levels of BCL2, BCL2A1 and BCL-xL. Our results also showed that pharmacologic inhibition of the activity of ABCG2, using fumitremorgin C and glefanine, significantly increased the chemosensitivity of BJAB and DOHH2 cells in the presence of stromal cells. However, this effect was not seen in the absence of stromal cells. Collectively, our data confirm that the stromal cells plays a role in inducing chemoresistance in DLBCL and that this effect is in part mediated by inducing high expression of drug protein transporters and antiapoptotic proteins. Inhibition of HH signaling as well as inhibition of drug transporters abrogates the stroma-induced chemoresistance suggesting that targeting these molecules may have a therapeutic value for the treatment of DLBCL. Disclosures:No relevant conflicts of interest to declare.

The Epstein-Barr Virus (EBV) Encoded Oncoprotein LMP1 Mediates Down Regulation of Shelterin Proteins, Formation of Telomere Aggregates and Multinuclearity.

AbstractAbstract 3625 Introduction:Tumour cells in classical Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL) are derived from germinal center B-cells and may or may not harbour the EBV genome. In both, EBV-negative and EBV-associated HL, the 3D nuclear organization of telomeres marks the transition from mononuclear Hodgkin (H-cell) to diagnostic multinuclear Reed-Sternberg (RS-cell) cells. Moreover, transient expression of the EBV-encoded oncoprotein LMP1 in the HL cell lines L-428 and HD-MyZ significantly increases the percentage of RS cells. However, the molecular mechanisms through which this selective LMP1 oncoprotein expression promotes the transition from H- to RS-cells is still unknown. In order to answer this question we analyzed the 3D telomere dynamics and the expression of key shelterin proteins at the transcriptional, translational and topographical localization level in the EBV-negative Burkitt cell line BJAB, stably transfected with an inducible LMP1 system (tetracycline on/off system). To mimic the permanent LMP1 expression as observed in tumour cells of EBV-associated Hodgkin's lymphoma, we extended the period of LMP1 expression up to 21 days and examined key proteins of the shelterin complex, 3D telomere dynamics and formation of multinuclear cells in interphase nuclei of BJAB Burkitt's lymphoma cells at day 1, 3, 7, 10, 14 and 21. Statistical analysis was performed using nested or two-way analysis of variance. Results:Already at day 3 LMP1 induced substantial down regulation of the shelterin key-components TRF1, TRF2 and POT1 at the transcriptional and protein level. This down regulation was reversible after LMP1 suppression; in particular, suppression of LMP1 induction at day 7 returned shelterin-key components to the initial level of expression at day 14. Massive LMP1 mediated down regulation of shelterin proteins was also confirmed by immunofluorescence. At day 7, stable LMP1 expression led to a significant increase of 3D telomere aggregates, nuclear volume and polyploid cells, most of them multinucleated, when compared to non-LMP1 expressing BJAB controls (p < 0.02). These changes progressed over time and at day 21 LMP1 expressing BJAB cells were characterized by highly significant increase of 3D telomere aggregates (p = 0.0005) and increased nuclear volume (p < 0.0001), compared to the LMP1-suppressed control. Discussion: In an experimental setting mimicking the in vivo conditions of EBV-associated HL, i.e. a permanent LMP1 expression in the H-cells, the LMP1 oncoprotein deregulates shelterin-expression, leads to telomere aggregates and mediates multinuclearity. Our findings point to a shelterin/telomere related key function of LMP1 in Reed-Sternberg cell formation in EBV-associated Hodgkin's lymphoma and may explain the four fold increased risk to develop HL within a median incubation time of four years after symptomatic EBV infection. The LMP1-dependent mechanisms involved are probably unprotected telomeres (shelterin down regulation) favouring chromosomal rearrangements as a result of telomeric aggregate formation and ongoing bridge-fusion- bridge cycles which lead to disturbed cytokinesis and finally multinuclearity. Disclosures:No relevant conflicts of interest to declare.

Upregulated JAK/STAT Signaling Represents a Major Mode of Resistance to HDAC Inhibition In Lymphoma and Provides a Rationale for Novel Combination Therapy

AbstractAbstract 434 BackgroundHistone deacetylase inhibitors (HDACis) have demonstrated significant clinical activity in hematologic malignancies; however, single agent response rates have ranged between 20–50% with the duration of response often measured in months, suggesting that drug resistance is a major mode of failure. The pathways through which these agents work and the means by which tumors develop resistance to them are poorly understood. Combination therapy targeting multiple oncogenic pathways holds the promise to improve upon both the depth and durability of these responses.We investigated the mechanisms of inherent and acquired resistance to HDACis in a broad range of lymphomas. By detailing the molecular pathways implicated in resistance to HDACi, we sought to identify novel combinations of compounds that could overcome potential mechanisms that confer resistance. Methods and ResultsWe tested two separate HDACis, LBH589 and SAHA in 51 cell lines representing a wide range of lymphomas including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, and Hodgkin lymphoma. Gene expression array data was generated for all these cell lines. We then identified genes that were significantly associated with resistance to both LBH589 and SAHA (p<.01) and applied hierarchical clustering to identify the functional significance of these genes. Histology was not predictive of sensitivity to either HDACi. These data were then analyzed using gene set enrichment to identify known molecular pathways associated with resistance. Activation of JAK/STAT signaling was found to be a major determinant of resistance among the cell lines that were relatively resistant to HDACi. (P<0.001, FDR <.25).To determine whether these genes that we found to be associated with resistance reflected potential mechanisms of acquired resistance to HDACi therapy, we separately engineered resistance to LBH589 and SAHA in three DLBCL cell lines (LY3, BJAB, Farage) through incremental dose escalation over a period of up to 6 months. Each of these three cell lines demonstrated sustained growth at drug concentrations that were at or above their original IC50. Each of these cell lines were then exposed to the other HDACi and tested for cross resistance. In each case, the cell lines demonstrated complete cross-resistance to the other drug. We then profiled the gene expression of these cell lines that had acquired resistance. Similar to our previous results, these cell lines demonstrated increased signaling through the JAK/STAT pathway, suggesting that mechanisms of inherent and acquired resistance are similar.We therefore reasoned that combining HDAC and JAK inhibition may overcome both inherent and acquired resistance. To investigate this hypothesis, we tested LBH589 and INCB018424, a JAK1/2 inhibitor, alone and in combination in the LY3, TMD-8, U2932, and BJAB cell lines. While INCB018424 demonstrated no single agent cytotoxicity, it yielded a high degree of synergy when combined with LBH589 with the combination index computed by the Chou-Talalay method ranging from .19 to .9. ConclusionHDACis show single agent activity in the treatment of a number of hematologic malignancies, however most patients develop resistance to these drugs after relatively short-lived remissions. Thus, the greatest promise of these drugs may lie in combination with other agents that target molecular pathways that underlie resistance to these drugs. Using gene expression profiling of a broad range of tumor types and sensitivity to HDACis we were able to identify activation of the JAK/STAT pathway as a common feature of inherent and acquired resistance to HDACis. We combined the JAK1/2 inhibitor INCB018424 with LBH589 and demonstrated a high degree of synergy. As the number of small molecule inhibitors with clinical activity increases, the need to identify rational preclinical combinations becomes greater. Pairing gene expression profiling and resistant cell lines is a promising approach to the selection of combinations likely to maximize clinical benefit while limiting toxicity. Disclosures:No relevant conflicts of interest to declare.

Exploratory Study on the Effects of Biodegradable Nanoparticles with Drugs on Malignant B Cells and on a Human/Mouse Model of Burkitt Lymphoma

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BNPs were non toxic for healthy animals, and had prolonged survival in this mice model of human lymphoma.

Involvement of Human Herpesvirus-6 Variant B in Classic Hodgkin's Lymphoma via DR7 Oncoprotein

Hodgkin's lymphoma (HL) is associated with the presence of EBV in Reed-Sternberg (RS) cells in ∼40% of cases. Here, we studied the presence of human herpesvirus type 6 (HHV-6) variant B in RS cells of HL patients and correlated results with clinical parameters. We then examined the implication of HHV-6 DR7B protein in cell deregulation. HHV-6 DR7B protein was produced in a Semliki Forest virus system. Polyclonal antibodies were then generated and used for immunochemical HHV-6 localization in HL biopsies. Binding between DR7B and p53 was studied using a double-hybrid system. Transactivation of NFκB was observed after transient transfection using reporter gene assays. We looked for Id2 factor expression after stable transfection of the BJAB cell line by reverse transcription-PCR and Western blot analysis. HHV-6 was more common in nodular sclerosis subtype HL, and DR7B oncoprotein was detected in RS cells for 73.7% of EBV-negative patients. Colocalization of EBV and HHV-6 was observed in RS cells of doubly infected patients. DR7B protein bound to human p53 protein. p105-p50/p65 mRNA expression and activation of the NFκB complex were increased when DR7B was expressed. Stable expression of DR7B exhibited a strong and uniform expression of Id2. A slightly higher percentage of remission was observed in patients with RS cells testing positive for DR7B than in those testing negative. Collectively, these data provide evidence for the implication of a novel agent, HHV-6, in cases of nodular sclerosis HL.

In vivo anti-lymphoma activity of an agonistic human recombinant anti-TRAIL-R2 minibody

A new single-chain fragment variable (scFv) to TRAIL-R2 receptor produced as minibody (MB2.23) was characterized for anti-lymphoma activity in vivo. For this purpose, a disseminated lymphoma model was generated by intraperitoneal inoculation of BJAB cells in severe combined immunodeficiency mice. Two weekly injections with MB2.23 (10mg/kg) were able to significantly increase the median survival time of lymphoma-bearing animals with respect to the vehicle-treated control mice, providing a rationale for further investigating the use of MB2.23 in anticancer therapy.