SNS01-T Exhibits Significant Anti-Tumoral Activity In Models Of Multiple Myeloma and Non-Hodgkins B Cell Lymphoma and Induces Cell Death In Malignant But Not Normal B Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 880-880
Author(s):  
Catherine A Taylor ◽  
Terence Tang ◽  
Sarah Francis ◽  
Zhongda Liu ◽  
Qifa Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Tumor Volume ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Rpmi 8226

Abstract SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and non-Hodgkins B-cell lymphomas. SNS01-T comprises a plasmid DNA (pExp5A) encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an eIF5A siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL). SNS01-T has demonstrated activity in MM xenograft models as well as in B cell lymphoma models of MCL and DLBCL, when administered twice weekly at doses ≥ 0.18 mg(nucleic acid)/kg. In this study we compared the ability of SNS01-T to transfect, regulate eIF5A expression, and kill MM, DLBCL, and MCL cell lines. Furthermore, the activity of SNS01-T in normal B cells was investigated. A previous study using a KAS-6/1 MM xenograft model demonstrated that the eIF5A siRNA and plasmid pExp5A both have anti-tumoral activity in MM but had a greater impact on tumour growth when combined together as SNS01-T. This finding was confirmed in this study in a second MM model (RPMI 8226) as well as in a DLBCL xenograft model. To determine the efficiency of SNS01-T transfection into malignant or normal B cells, the pExp5A plasmid and eIF5A siRNA were labeled with FITC and DY547, respectively, packaged into nanoparticles using polyethylenimine polymer, and used to transfect cultured cells. FACS analysis was used to determine the percent of the cell population transfected with plasmid, siRNA, or both. RT-qPCR was used to assess biological activity of SNS01-T by quantifying the expression of eIF5AK50R mRNA transgene and endogenous eIF5A mRNA in a variety of B cell lines. The IC50 of SNS01-T in a panel of MM, MCL, and DLBCL cell lines was determined by XTT assay. SCID mice bearing either RPMI 8226 MM tumours or SuDHL6 GCB DLBCL tumours were treated with pExp5A plasmid (formulated with PEI and control siRNA), eIF5A siRNA (formulated with PEI and a control plasmid), or SNS01-T at 0.375 mg/kg twice per week by intravenous injection. SNS01-T was able to transfect MM, MCL, and DLBCL cell lines, although the proportion of cells transfected with both plasmid and siRNA was higher in MM cells. Transfection of SNS01-T resulted in expression of the transgene as well as a statistically significant reduction in expression of eIF5A mRNA compared to untreated controls for all three cell types. In contrast, normal B cells were found to take up fluorescently-labeled SNS01-T with reduced efficiency compared to RPMI 8226 MM cells. Futhermore, SNS01-T was observed to induce cell death in RPMI 8226 MM cells but not in normal B cells. In the RPMI 8226 xenograft model, treatment with either the pExp5A plasmid alone or eIF5A siRNA alone resulted in a 66 % reduction (p < 0.0001) or 44 % reduction (p < 0.05) in tumor volume compared to the control group at day 24 of the study. In contrast, treatment with SNS01-T, which contains both the pExp5A plasmid and the eIF5A siRNA, resulted in an 86 % (p < 0.0001) reduction in tumor volume. A similar result was observed in the SuDHL6 model with a 14 % reduction or 27 % reduction (p < 0.05) in tumor volume compared to the control group at day 20 of the study following treatment with pExp5A plasmid or eIF5A siRNA, respectively. In contrast, treatment with SNS01-T resulted in a 79 % (p < 0.0001) reduction in tumor volume. Collectively, these preclinical studies indicate that SNS01-T therapy has significant potential against MM, MCL, and DLBCL. Disclosures: Taylor: Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Micro-RNA Gene Deregulation by Chromosome Translocations with Fragile Genomic Regions in B-Cell Lymphoma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 786-786
Author(s):  
Bjoern Schneider ◽  
Stefan Nagel ◽  
Maren Kaufmann ◽  
Hans G. Drexler ◽  
Roderick A.F. MacLeod
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Micro Rna ◽  
Lymphoma Cell ◽  
Chromosome Rearrangements ◽  
Causal Connection

Abstract Micro-RNA (miR) genes posttranscriptionally modulate target gene expression via imperfect 3′-UTR matching sequences and play key roles in development, homeostasis and cancer. Little is known how miR genes are themselves regulated, or deregulated in cancer. Chief paradigm for neoplastic miR deregulation concerns miR-17/92 cluster members subject to genomic amplification in B-cell lymphoma. While the repeated occurrence of oncogenic miR genes at or near chromosomal breakpoints in cancer links chromosome fragility to oncogenic miR deregulation, direct evidence of a causal connection remains tenuous. We found that t(3;7)(q27;q32) in a B-cell lymphoma cell line joins 5′-BCL6 to a noncoding region of chromosome 7 inside a common chromosomal fragile site (FRA7H). In these cells hybrid mRNA was absent, unlike canonical BCL6 translocations which involve promoter exchange yielding hybrid mRNA. Affected cells instead showed downregulation of miR-29b-1, the only gene located within FRA7H - a recurrent transcriptional feature of B-cell lymphoma subsets. In another BCL6 translocation, t(3;13)(q27;q31)t(13;12)(q31;p11), which 5′-RACE also showed to be non-fusogenic, long distance inverse (LDI)-PCR revealed junction of 5′-BCL6 to chromosome 13 sequences inside the miR-17/92 host gene MIRH1 (alias c13orf25). FISH using a sensitive tyramide amplification protocol with c13orf25 clones confirmed the presence of a cryptic BCL6-MIRH1 rearrangement. Surprisingly, reverse transcriptase quantitative (q) PCR assay revealed weak MIRH1 expression using 3′-primers. In contrast, repeating the assay using more central primers covering the miR-17/92 coding region showed massive upregulation. 3′-RACE confirmed a novel high level MIRH1 transcript truncated by 3.1 kbp. Quantitative genomic PCR and FISH excluded miR-17/92 genomic copy number alteration, while LDI-PCR analysis showed that formation of truncated MIRH1 involved multiple DNA cuts at 3q27 (x1), 12p11 (x1), and 13q31 (x5) – the last including a complex excision/inversion/insertion rearrangement. Stress induced DNA duplex destabilization (SIDD) analysis revealed that 6 of 7 breaks precisely coincided with fragility peaks. Taken together, these data suggest a novel role for BCL6 translocations in the deregulation of miR genes near sites of chromosome or DNA instability. BCL6 has been shown to suppress p53 in germinal center B-cells thus protecting B-cells from apoptosis induced by DNA damage, offering a possible explanation for chromosome rearrangements associated with genomic fragility therein. Chromosomal MIRH1 dysregulation is not limited to BCL6 expressing lymphomas, however: cytogenetic investigations performed on diverse leukemia-lymphoma cell lines, including those derived from multiple myeloma and plasma cell leukemia, showed 11/50 with cytogenetic rearrangements at or near MIRH1. In sister cell lines sequentially established at diagnosis and relapse of multiple myeloma, only the latter showed miR-17/92 chromosomal rearrangement and upregulation. Interestingly miR overexpression was limited to miR-92, while miR-17/18 were barely expressed. FISH analysis and qPCR showed that discrepant expression was associated with rearrangement upstream of MIRH1. In brief, our data show that like other cancer genes, oncogenic miRs are subject to dysregulation mediated by structural chromosome rearrangements.

Aberrant Somatic Hypermutation Targets an Extensive Set of Genes in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1528-1528 ◽  
Author(s):  
Laura Pasqualucci ◽  
Roberta Guglielmino ◽  
Sami N. Malek ◽  
Urban Novak ◽  
Mara Compagno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Total N ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Genomic instability is a driving force in tumor development that can be achieved by a variety of mechanisms, such as defective chromosome segregation or inactivation of the DNA mismatch repair pathway. Although B-cell lymphomas are associated with chromosomal translocations deregulating oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has long not been described. We have reported that the somatic hypermutation process (SHM), which normally targets the immunoglobulin variable region (IgV) and BCL6 genes in germinal center (GC) B-cells, functions aberrantly in >50% of diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (Pasqualucci et al., Nature412:341, 2001). As a consequence, multiple somatic mutations are introduced into the 5′ region of genes that do not represent physiologic SHM targets, including known proto-oncogenes such as PIM1, PAX5, RhoH/TTF and cMYC. To further define the extent of this phenomenon, termed aberrant somatic hypermutation (ASHM), and to identify additional hypermutated loci of possible pathogenetic significance in DLBCL, we screened 113 genes for the presence of mutations affecting their 5′ sequences (≥1.3 Kb from the transcription start site, the target region for SHM) in 10 DLBCL cell lines. Fifteen genes (13.3%) were found to harbor a significant number of mutations (p<0.05), with 70% of the cell lines being mutated in 7 or more genes; among these, six B-cell specific loci -BCL7A, CIITA, IRF4, LRMP, NCOA3 and SIAT1- carried 9–53 mutational events distributed in 20 to 70% of the cases, corresponding to an overall mutation frequency of 0.032–0.15% (frequency in the mutated cases: 0.07–0.25%). The same genes were found hypermutated in a panel of 20 primary DLBCL biopsies, which displayed an overall mutation load of 7 to 45 distinct events/gene (total N=125). Mutations were of somatic origin, independent of chromosomal translocations to the Ig loci and were restricted to the first 1.5–2 Kb from the promoter. In addition, analogous to previously identified SHM and ASHM targets, the mutations exhibited characteristic features, including a bias for transitions over transversions, preferential hotspot (RGYW/WRCY motifs) targeting, and higher frequencies at G:C pairs. However, in contrast to physiologic SHM targets such as IgV and BCL6, none of the 4 newly identified hypermutated genes that have been analyzed so far (BCL7A, CIITA, SIAT1, LRMP) displayed significant levels of mutations in purified normal GC B-cells as well as in other B-cell malignancies. This finding indicates that these genes represent aberrant hypermutation targets resulting from a tumor-associated malfunction, possibly a loss of target specificity of the physiologic SHM process. Considering previous results and the present survey, 17 (13%) out of 130 genes investigated have been found involved in ASHM, suggesting that this aberrant activity may involve an extensive set of target genes in DLBCL. Since the mutations affect both regulatory and coding sequences of the targeted genes, aberrant SHM may represent a major contributor to the pathogenesis of this disease and may explain in part its phenotypic and clinical heterogeneity.

Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5023-5023
Author(s):  
Susana Hernández-García ◽  
Mercè de Frias ◽  
Clara Campàs ◽  
Bruno Paiva ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Hematologic Malignancy ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Mutational Status

Abstract Abstract 5023 Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes. Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients. We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status. Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient. Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

scholarly journals Insulin-like Growth Factor-1 Receptor (IGF-1R) As a Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4828-4828
Author(s):  
Xiangxiang Zhou ◽  
Lingyun Geng ◽  
Xinyu Li ◽  
Peipei Li ◽  
Kang Lu ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Caspase 3 ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Protein Levels ◽  
Proliferation And Apoptosis ◽  
Dose Dependent Decrease

Abstract Introduction : The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is dysregulated in various tumor entities and hematological malignancies including chronic lymphocytic leukemia and mantle cell lymphoma. The implication of IGF1R in the development and progression of cancer has led to its current evaluation in clinical trials as a potential therapeutic target for solid tumors. However, its functional significance in diffuse large B-cell lymphoma (DLBCL) remains poorly characterized. We hypothesized that IGF1R plays a key role in the pathogenesis and progression of DLBCL. In this present study, we evaluated the expression and function of IGF1R in both B cell lines and DLBCL tissues, as well as assessed the proliferation and apoptosis of DLBCL cells when treated with IGF-1R inhibitor, AG1024. Methods : Expression of IGF1R in B-cell lymphoma cell lines (LY1, LY8, Mino, Jeko-1, and SP53) was evaluated by Western blotting. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Blood samples and araffin-embedded tissues from 30 initial-diagnosed DLBCL patients prior to therapeutic interventions as a study group, and from 15 patients with reactive hyperplasia lymphnode as a control group were collected with informed consents. Immunohistochemisty (IHC) was conducted to assess the expression of IGF-1R in lymphoma tissues. Correlations between IGF1R expression and the clinical characteristics of DLBCL patients were further analyzed. DLBCL cell lines (LY1 and LY8) were treated with an IGF1R specific small molecular inhibitor, AG1024, cell proliferation was analyzed by cell counting kit (CCK-8). Effects of inhibitor or stimulator on the apoptosis of LY1 and LY8 cells were assessed by Annexin-V/PI and Annexin-V/7AAD, respectively. Expression of apoptosis-related protein, including Caspase-3 and Mcl-1, was evaluated by western blotting. Protein levels of downstream targets of IGF-1 signaling were also detected. Results : Significantly upregulation of both phoaphprylated and total IGF1R protein levels were found in B-cell lymphoma cells (LY1, LY8, Mino, Jeko-1 and SP53) (Fig 1.A). IHC was conducted and revealed significantly enhancement of IGF1R expression in DLBCL patients (Fig 1.B). Among the included DLBCL patients and control group with inreactive hyperplastic lymphadenitis, the positive rate of IGF1R was 90% and 20%, respectively. We then investigated the function of IGF1R inhibitors on the proliferation and apoptosis of DLBCL cells. LY8 cells were treated with different doses of AG1024 at 24-96 hours. Cell proliferation was inhibited by 60% when treated with AG1024 at the concentration of 15µM for 72-hours (Fig 1.C). Culture of LY1 and LY8 cells in the presence of 10µM and 15µM AG1024 concentration for 24-hours resulted in 13% (p<0.05) and 33% (p<0.001) cell apoptosis, respectively (Fig 1.D). Inhibition of IGF1R by AG1024 also resulted in induction of cleaved-Caspase-3, as well as reduction of Mcl-1(Fig 1.E-F). In order to investigate the mechanisms involved in the dysregultaion of IGF1R in DLBCL, LY8 cells were treated with 5 to 15 µM AG1024, the results revealed that AG1024 caused a dose-dependent decrease in the levels of phosphorylated IGF1R, AKT and ERK (Fig 1.G). Treatment of LY8 cells with recombinant human IGF-1 led to enhanced phosporylation levels of IGF1R, AKT and ERK (Fig 1.H). Conclusion s: Our investigation observed that expression levels of IGF-1R were up-regulated in both B-cell lymphoma cells and DLBCL tissues. DLBCL cells treated with IGF-1R inhibitor, AG1024, revealed reduced proliferation and increased apoptosis rate. In addition, induction of cleaved-Caspase-3 was also found in LY1 treated with AG1024. AG1024 caused a dose-dependent decrease in the phosphorylation levels of IGF1R, AKT and ERK. This study suggests that IGF1R could be a potential molecular target for the treatment of DLBCL. The IGF-1R inhibitor is a promising therapeutic approach for DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Klotho/IGF-1R Regulates Tumor Growth and Predicts Prognosis in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2924-2924
Author(s):  
Xiangxiang Zhou ◽  
Ying Li ◽  
Xinyu Li ◽  
Lingyun Geng ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Klotho Protein ◽  
Xenograft Models

Abstract Introduction: Klotho is an anti-aging gene with an extracellular domain. Mice with Klotho knockout exhibited obvious impairment in B-cell development. Evolving evidence indicates that Klotho modulates the proliferation and survival via targeting insulin-like growth factor-1 receptor (IGF-1R) in several cancers. However, the expression and biological role of Klotho in B-cell non-Hodgkin lymphoma (B-NHL) has not been elucidated to date. We hypothesized that Klotho could modulate the tumor growth and predicts prognosis in diffuse large B-cell lymphoma (DLBCL) through inhibiting IGF-1R activation. The aim of this study is to characterize the functional significance of Klotho and the therapeutic potential of its secreted form in DLBCL. Methods: Lymph nodes samples from 50 de novo DLBCL and 20 reactive hyperplasia cases were collected with informed consents. Klotho expression were assessed by Immunohistochemistry. CD19+ B-cells and peripheral blood mononuclear cells were isolated with informed consents from healthy donors. Expression levels of Klotho mRNA and protein in DLBCL cells were determined by quantitative RT-PCR and western blotting. Lentivirus vectors either encoding Klotho (LV-KL) or empty lentiviral vector (LV-Con) were stably transfected into DLBCL cells. Cell viability and apoptosis were analyzed by cell counting kit-8 and Annexin V-PE/7AAD staining. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. SCID-Beige mice were subcutaneously injected with DLBCL cells to establish xenograft model. Results: We observed markedly decreased level of Klotho protein in DLBCL lymph nodes (Fig. 1A). Expression of Klotho protein exhibited significantly negative correlation with Ann Arbor stage of DLBCL patients (p=0.002). Level of Klotho protein was negatively correlates with the media overall survival (OS), suggesting lower Klotho expression is associated with poor OS in DLBCL ((Fig. 1B, p=0.045). Reduction of Klotho was also confirmed in DLBCL cell lines at mRNA and protein level (Fig. 1C). We next functionally interrogated the role of Klotho in DLBCL cell lines and xenograft models. Stably expression of LV-KL in DLBCL cell lines resulted in dramatically decreased cell proliferation and incremental apoptotic rates when compared to LV-Con (Fig. 2A and B). We validated the changed expression of critical targets known to govern apoptosis in DLBCL cells transfected with LV-KL. Xenograft models with Klotho overexpression revealed significantly abrogated tumor growth compared to control group (Fig. 2C). Interestingly, lower levels of Ki67 were observed in mice treated with LV-KL (Fig. 2D). These results highlighted the proliferation-inhibitory and apoptosis-inductive activities of Klotho in DLBCL cells. The underlying mechanism driving the tumor suppressive potential of Klotho was investigated. Surprisingly, we observed that the Klotho-induced inhibition of cell viability was only fewer restored by IGF-1 in DLBCL cells transfected with LV-KL (Fig. 3A). Reductive phosphorylation of IGF-1R and its downstream targets (AKT and ERK1/2) were observed in DLBCL cells with Klotho overexpression (Fig. 3B). In addition, we evaluated the regulation of Klotho on IGF-1R signaling in vivo. Decreased phosphrolation of IGF-1R as well as its downstream targets were observed in mice treated with LV-KL compared to the control group (Fig. 3C). Lastly, we explored the activity of secreted Klotho protein (rhKL). The rhKL was found to be active in vitro and significantly reduced the viabilities of DLBCL cells (Fig. 3D). Moreover, combination with rhKL increased the sensitivity of DLBCL cells to adriamycin. The in vivo activity of rhKL in DLBCL xenograft model was also detected. Significantly decreased tumor volumes were noted in mice treated with rhKL compared with those treated with vehicle control (Fig. 3E). Moreover, reductive expression level of Ki67 was observed in rhKL-treated group (Fig. 3F). Conclusions: Our observations identified for the first time that loss of Klotho expression contributed to the development and poor prognosis via activating IGF-1R in DLBCL. Given the in vivo tumor suppressive activity of secreted Klotho protein, it may serve as a potential strategy for the development of novel therapeutic interventions for DLBCL. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals RNA Sequencing of Hodgkin Lymphoma Reed-Sternberg Cells Uncovers a Plasma Cell Signature and Escape from NK Cell Recognition

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 549-549 ◽  
Author(s):  
Ethel Cesarman ◽  
Mikhail Roshal ◽  
Jonathan Reichel ◽  
Wagner Florian ◽  
Bhavneet Binder ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Plasma Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Provision Of Services ◽  
Slam Family

Introduction: Previous gene expression profiling studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines and microdissected HRS cells from tissue biopsies given the difficulty of isolating sparse Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. We previously used flow sorting to separate HRS cells from fresh or viably frozen cHL biopsies, and performed the first full exome sequencing of HRS cells. Here we report use of the same cell separation approach to examine the HRS cell transcriptome using RNA sequencing. Methods: We used flow cytometric cell sorting and low-input RNA sequencing to generate full transcriptome data from viable primary HRS cells, along with intratumor B cells. Nine primary cases of cHL and four cell lines were assessed for RNA expression, expressed mutations, cell type of origin, signaling pathways, gene fusions and pathogen identification. We used immunohistochemistry to evaluate expression of PDIA6 and CD48 in the 9 cases sequenced and a tissue microarray containing 16 additional cases of cHL. Flow cytometry for CD48 was performed in two cell lines and 5 primary cases. Results: Clustering show that primary HRS cells have a transcriptional profile that is unique, and different from that of intratumoral B cells, as well as cHL cell lines. Comparison of HRS cells with normal cellular subsets indicated plasma cell differentiation, suggesting that the cell of origin is a B cell on its way to becoming a plasma cell. Clustering with B cells showed much lower similarity. Consistent with plasma cell differentiation, we uncovered an unfolded protein response UPR) signature, shared with plasma cell neoplasms and, to a lesser extent, activated B cell (ABC) diffuse large B cell lymphoma, but not other B cell lymphoma types, including primary mediastinal B cell lymphoma (PMBCL). Among other UPR response genes, PDIA6 showed strong downregulation at the RNA level (2.4 logFC, p=9.4E-17). This finding was validated by immunohistochemistry for PDIA6, which showed strong positivity in the HRS cells of all 25 cases examined, confirming that this is a common feature of cHL, including nodular sclerosis and mixed cellularity subtypes. Top upregulated genes included those involved in oncogenesis (HGF/MET, NFkB/apoptosis inhibition), stem cell differentiation (homeobox genes MEIS1 and PBX1), and mitotic checkpoints, mitotic spindle formation and DNA repair, possibly explaining the unique nuclear morphology of HRS cells. Downregulation of MHC-1 and MHC-2 driven antigen processing and presentation was confirmed, and so was overexpression of PDL1 (CD274). Importantly, we detected loss of SLAM family receptors, which serve as activation signals for NK cells providing an additional mechanism for tumor immune evasion. One of these is CD48 (-2.63 logFC, p=1.56E-05), which was confirmed to be strongly downregulated. This finding was confirmed by immunohistochemistry (25 cases) and flow cytometry (2 cell lines and 5 primary cases) on the expanded sample set. Given that only some cHL cases are associated with EBV infection, it has been speculated that other viruses are involved in negative cases. However, our analysis did not reveal additional viruses in the HRS cells. Conclusions: Our data indicate that cHL more closely resembles plasma cells than B cells, and plasma cell malignancies than other lymphomas. The salient feature of plasmacytic differentiation is a UPR, which is seen in HRS cells and multiple myeloma. In contrast, UPR is not a feature of primary mediastinal B cell lymphoma, which is thought to be the DLBCL most similar to cHL clinically, immunophenotypically and in terms of gene expression patterns. We also provide an integrated view of potential immune evasion mechanisms by HRS cells that potentially explain lack of anti-tumor T or innate response. These include lack of antigen presentation due to B2M mutations, overexpression of PDL1 and PDL2, immunosuppressive cytokine secretion and, for the first time, a demonstration of lack of NK activating receptors of the SLAM family. Lack of SLAM family receptors may explain lack of NK cells clearance of HRS cells in the face of MHC-I downregulation. It has long been suspected that cHL is a tumor where there likely exists a previously undiscovered virus in addition to EBV, but RNA sequencing failed to reveal additional infectious transcripts in the HRS cells. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Brody:Kite Pharma: Research Funding; Celldex Therapeutics: Research Funding; Genentech: Research Funding; Acerta Pharma: Research Funding; Oncovir, Inc.: Research Funding; BMS: Research Funding; Merck: Research Funding. Dave:Data Driven Bioscience: Equity Ownership.

Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1401-1401 ◽  
Author(s):  
Massimo Mangiola ◽  
Kate Welsh ◽  
Shinichi Kitada ◽  
Irene M. Pedersen ◽  
Nuzhat Pathan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
Efficient Induction ◽  
Anti Cd20

Abstract We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

Lenalidomide Efficacy in Activated B-Cell-Like Subtype Diffuse Large B-Cell Lymphoma Is Dependent Upon IRF4 and Cereblon Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3287-3287
Author(s):  
Ling-Hua Zhang ◽  
Jolanta Kosek ◽  
Maria Wang ◽  
Carla Heise ◽  
Peter H Schafer ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Employment Equity ◽  
Large B Cell Lymphoma ◽  
Equity Ownership

Abstract Abstract 3287 Background: Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods: A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results: Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells. This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p<0.05) downregulate IRF4 protein levels in sensitive cell lines such as OCI-Ly10 and U2932. IRF4 inhibition by lenalidomide reduced CARD11-BCL-10-MALT1 complex activity of ABC-DLBCL cells (as measured by BCL-10 cleavage) and resulted in downregulation of B-cell receptor (BCR)-dependent NF-κB activity. An NF-κB-driven luciferase assay revealed that lenalidomide (1 μM) inhibited transcriptional activity of NF-κB up to 56% in the sensitive ABC-DLBCL cell lines OCI-Ly10 (p <0.05) and U2932 (p <0.01) after 2-day drug treatment. Lenalidomide also significantly (p <0.05) inhibited DNA binding by Rel A/p65, p50 and c-rel/p70 in 4 lines of ABC cells. While IRF4-specific siRNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression conferred cell resistance to lenalidomide, indicating the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC DLBCL. Furthermore, knockdown of CRBN in OCI-Ly10 (p <0.05) and U2932 (p <0.01) conferred resistance to lenalidomide as demonstrated by the abrogation of the inhibitory effects of lenalidomide on IRF4 expression, BCL-10 cleavage, NF-κB activity, and proliferation of these cells, whereas the activity of inhibitors to PKC β and IKKα/β (LY-333,531 and CC-415501, respectively) remained unaffected. These data indicate that antitumor effects of lenalidomide on ABC-DLBCL cells require the presence of cereblon. Conclusions: These data may provide a mechanism for the preferential efficacy of lenalidomide in ABC-DLBCL observed in clinical studies. These findings suggest that lenalidomide has direct antitumor activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signaling pathway in a cereblon-dependent manner (also see Figure below). Disclosures: Zhang: Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Heise:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

Combination Therapy with Novel Nanoparticle, SNS01-T, and Lenalidomide Triggers Synergistic Cytotoxicity in Vitro and in Vivo in Multiple Myeloma and Mantle Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4223-4223
Author(s):  
Catherine A Taylor ◽  
Terence Tang ◽  
Zhongda Liu ◽  
Sarah Francis ◽  
Zheng Qifa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Significant Activity ◽  
Rpmi 8226

Abstract Abstract 4223 SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and B-cell lymphomas. SNS01-T is comprised of a plasmid encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM). SNS01-T and its preclinical precursors have been studied extensively in multiple myeloma and B cell lymphoma tumor models. In this study we tested the in vitro and in vivo anti-cancer activity of SNS01-T in combination with the immunomodulatory drug lenalidomide. The combination of low doses of SNS01-T and lenalidomide synergistically reduced viability of RPMI 8226 MM cells and induced apoptosis to a greater degree than either drug alone. To determine whether SNS01-T treatment increases the anti-myeloma activity of lenalidomide in vivo, 0.375 mg/kg SNS01-T was combined with either 15 or 50 mg/kg lenalidomide in a RPMI 8226 xenograft model of multiple myeloma. Mice were dosed for two cycles of treatment for a total of 11 weeks of dosing. Mice with no measurable tumor at the end of the first cycle of treatment did not receive treatment in the second cycle but were monitored closely for tumor recurrence. A two-week observation period at the end of the study allowed monitoring of tumor growth after the cessation of the second cycle of treatment. At the end of the second cycle of dosing, tumor growth was inhibited by 84 % (p < 0.0001), 34 % (p = 0.05), and 98.1 % (p << 0.0001) in animals treated with SNS01-T, 50 mg/kg lenalidomide, and SNS01-T plus 50 mg/kg lenalidomide, respectively. Complete tumor regression (undetectable tumor) was achieved in 40% of mice treated with SNS01-T, 0% of mice treated with 50 mg/kg lenalidomide, and 83% of mice treated with the combination therapy of SNS01-T and 50 mg/kg lenalidomide. Complete regression of tumors treated with the combination therapy was maintained for more than 8 weeks without treatment until the end of the study in 4 of 6 (67%) of treated mice. Combining SNS01-T treatment with 50 mg/kg lenalidomide inhibited tumor growth more effectively than either drug alone and prolonged survival with 100% of mice surviving to the end of the 102-day study. Combination therapy with SNS01-T and 15 mg/kg lenalidomide also demonstrated significant activity in a murine JMV-2 mantle cell lymphoma (MCL) xenograft model. Treatment of mice with the drug combination of SNS01-T and lenalidomide resulted in a statistically significant increase in survival compared to either SNS01-T (p = 0.002; logrank test) or lenalidomide (p = 0.007) alone. Collectively, these preclinical studies indicate that the combination therapy of SNS01-T and lenalidomide is well tolerated, has significant activity against MM and MCL, and provides a strong rationale to evaluate SNS01-T and lenalidomide combination therapy to improve patient outcome in MM and B cell lymphomas. Disclosures: Taylor: Senesco Technologies Inc.: stock options Other. Dondero:Senesco Technologies Inc.: Employment. Thompson:Senesco Technologies Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Inactivation Of BANK1 By a Novel IGH-Associated Translocation and 5’ Hypermethylation In B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2497-2497 ◽  
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Jiong Yan ◽  
Leonardo Boiocchi ◽  
Shuhua Cheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Methylation Status ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Intron 1 ◽  
Large B Cell

Abstract A novel IGH-associated reciprocal translocation, t(4;14)(q24;q32), was identified, along with trisomy 9, in 20 of 20 metaphases by conventional karyotyping in a case of malignant gastric post-transplant lymphoproliferative disorder (PTLD). Cloning of the translocation site by inverse PCR identified BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific adaptor protein with putative functions in B-cell receptor and CD40 signaling, as a novel IGH translocation partner. The breakpoints were located at the Sα region of IGH and intron 1 of BANK1. The translocation juxtaposed the two genes in opposite orientations, and surprisingly, resulted in transcriptional inactivation of BANK1 as a result of dissociation of the major BANK1 promoter. While BANK1 isoforms were expressed in all tonsillar B-cells, with lower levels (∼ 5 fold) in the germinal centers (GC) compared to naïve and memory B-cells, transcription from the major promoter in the tumor was absent and transcription from the minor promoter was reduced 50% relative to GC B-cells, suggesting that the non-translocated BANK1 allele was also inactivated. The total BANK1 expression was very low (∼10% of normal GC B cells) and crytic promoter activation was not identified. Several genes (PPP3CA, MIR1255A, FLJ20021 and SLC39A8), located 180 to 440 kb away from BANK1, were analyzed for mRNA expression; there is no significant activation in any of these genes, further supporting that BANK1is indeed the target gene affected by the translocation. Interphase FISH using break-apart BANK1 probes confirmed breakpoint in the index case but did not identify translocations in additional 15 PTLDs and 68 diffuse large B-cell lymphomas (DLBCL), implying that BANK1 translocation may be a rare event. To determine if BANK1 inactivation may occur in B-cell lymphomas by other mechanisms, 23 B-cell lymphoma cell lines, including 8 Burkitt lymphoma (BL), 9 diffuse large B cell lymphoma (DLBCL), 3 primary effusion lymphoma (PEL), and 3 classical Hodgkin lymphoma (cHL) were bisulfite sequenced to assess the methylation status of 37 CpG dinucleotides in a 436 base-pair region at the 5’ end of BANK1, which extends across exon 1 into the 5’ portion of intron 1. High level of methylation (>60% methylation on average among all CpGs) was seen in all 3 cHL and 2 of 3 PEL cell lines. Regional methylation was seen in 3 of 8 BL lines and 1 of 3 PEL lines. No hypermemethylation was identified in the DLBCL lines or in normal tonsils. Hypermethylation was associated with almost complete silencing of BANK1 transcription. In the DLBCL lines and BL lines without BANK1 hypermethylation, BANK1mRNA expressions were variable, ranging from <5% to 130% of GCB cells. To confirm that BANK1 hypermethylation is present in primary lymphoma cases, methylation status of 17 of the 37 CpGs were assessed in 23 cHL cases using en bloc formalin-fixed, paraffin-embedded materials and also laser-capture micro-issected Hodgkin/Reed-Sternberg (HRS) cells. There was evidence of BANK1 hypermethylation in the tumor cells in 9 of 23 cHL. Tumor cell specificity of BANK1 hypermethylation was further confirmed in 4 cHL cases using micro-dissected HRS cells. HRS cells were negative for BANK1 in 28 of 29 cHL cases examined by immunohistochemistry, suggesting that other mechanisms other than DNA methylation may be responsible for silencing BANK1expression. To investigate whether BANK1 has biological effects on B-cells related to lymphoma development, exogenous BANK1 was re-introduced to BC3, a PEL cell line showing marked BANK1 hypermethylation with absence of BANK1 expression. We established a stable doxycycline-inducible BC3 cell line expressing BANK1. Inhibition of cell growth was observed 2 to 3 days after doxycyline induction, and the number of viable cells with transfected BANK1 was only 25% compared to BC3 cells carry vehicle alone at day 6. An analysis of 5-bromo-2’ deoxyuridine (BrdU) incorporation after 48 hours of doxycline induction revealed that the fraction of cells in S-phase was reduced by 50% in the BANK1 transfectants, suggesting that BANK1has a negative effect on cell proliferation in these B cells. In summary, we have identified a novel IGH translocation partner and provide an example of an unusual consequence (gene inactivation) of IGH-associated translocation. We provide for the first time evidence of a potential role of BANK1 down-regulation in the development of B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

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