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 A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3941-3941
Author(s):  
David M Goldenberg ◽  
Serengulam Govindan ◽  
Tom M Cardillo ◽  
Robert M Sharkey
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

scholarly journals Targeting ubiquitin-activating enzyme induces ER stress–mediated apoptosis in B-cell lymphoma cells

2019 ◽  
Vol 3 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Scott Best ◽  
Taylor Hashiguchi ◽  
Adam Kittai ◽  
Nur Bruss ◽  
Cody Paiva ◽  
...  
Keyword(s):  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Cell Of Origin ◽  
Protein Ubiquitination

Abstract Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)–like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243–induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

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

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

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.

Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 886-886 ◽  
Author(s):  
Lapo Alinari ◽  
Erin Hertlein ◽  
David M. Goldenberg ◽  
Rosa Lapalombella ◽  
Fengting Yan ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Preclinical Model ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals CD30 Promotes Proliferation Via ERK 1/2 and p38 MAPK Signal Pathways in ABC-Diffuse Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4337-4337
Author(s):  
Weimin Huang ◽  
Xiaolei Wei ◽  
Yongqiang Wei ◽  
Ru Feng
Keyword(s):  
B Cell ◽  
P38 Mapk ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
In Vivo Study ◽  
Signal Pathways ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background CD30 was expressed in about 40% diffuse large B cell lymphoma (DLBCL), epically in activated B cell subtype DLBCL(ABC-DLBCL) and implied superior outcome. however, the underlying mechanisms remains unclear. Methods Three ABC-DLBCL cell lines (SuDHL2, TMD8, HBL1) were used for construction of stably CD30 expression cell lines. The proliferation of these cell lines was measured by MTT assay. Cell cycles were detected by Flow Cytometry after staining with PI. Expression of CyclinD1, p27, CDK2, ERK, phosphorylation-ERK, p38, phosphorylation p38 was detected by Western Blot. Nude mice were used in vivo study. Results With CD30 overexpression, the proliferation and division of SuDHL2, TMD8 and HBL1 cell lines were promoted. CyclilnD1, CDK2 were upregulated and p27 was downregulated after of CD30 overexpression. Furthermore, CD30 was knock down by treated CD30 shRNA in CD30 overexpression cell lines. CD30 KD could reverse the the proliferation induced by CD30 expression. CD30 overexpression increased the phosphorylation of ERK and p38 in ABC-DLBCL cells. In vivo study, CD30 overexpression was correlated with cell proliferation and division. Conclusion Taken together, our date showed CD30 promotes proliferation and division by activating ERK 1/2 and p38 MAPK signal pathways, and targeting CD30 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in ABC-DLBCLs. Key words: CD30, ERK 1/2, p38 MAPK, ABC- diffuse large B cell lymphoma Disclosures No relevant conflicts of interest to declare.

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