Targeting Aurora Kinase in Aggressive B-Cell Non-Hodgkin's Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 284-284 ◽  
Author(s):  
Daruka Mahadevan ◽  
Wenqing Qi ◽  
Laurence Cooke ◽  
Xiabing Lui ◽  
Daniel Oscar Persky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
Parp Cleavage ◽  
Tumor Growth Inhibition ◽  
Aurora A ◽  
Aurora Kinases ◽  
Over Expression

Abstract Abstract 284 Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Aurora kinases during the cell cycle over-rides mitotic and spindle check points leading to aneuploidy in many human cancers; Aurora kinases are therefore attractive therapeutic targets. Gene expression profiling in aggressive B- and T-cell non-Hodgkin's lymphoma (NHL) has shown the Aurora kinases to be over-expressed and they may be key component genes of the ‘proliferative' signature. We hypothesized (1) Aurora kinases are over-expressed in human aggressive B-cell NHL (mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (TFL)), (2) Aurora ATP-binding site small molecule inhibitor (SMI) is effective in promoting apoptosis in cell culture and tumor growth inhibition (TGI) in mouse xenograft model(s) of NHL, and (3) Aurora SMI will be safe and effective in treating patients with relapsed aggressive B-cell NHL in early phase clinical trials. To analyze Aurora expression, tissue microarrays (TMA) were constructed from 43 patients with DLBCL and 40 with MCL, and Aurora A and B expression optimized with commercially available antibodies by immunohistochemistry (IHC). The IHC was rated as a staining intensity on a scale of 0 to 3+. The NHL TMAs demonstrated intense staining (2+ to 3+) for Aurora A (nucleus) and Aurora B (nucleus) in >60% compared to normal lymph nodes. We also analyzed the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) publicly available database for Aurora A and B expression in MCL and found a worse survival in those with A > B over-expression (p<0.01). Since both Auroras are transforming genes, the LLMPP data support the conclusion that these S/T kinases are associated with a poor prognosis and are potential targets for therapy. Western blotting analysis of 13 B-cell NHL cell lines (DLBCL, MCL and TFL) for Aurora A and B expression showed significant over-expression compared to B-cells isolated from normal lymph nodes. Aurora A knockdown by shRNA in the B-NHL cell lines showed inhibition of mitosis with a polyploid phenotype (4n, 8n) that ends in apoptosis as shown by PARP-cleavage. The Aurora A specific inhibitor (MLN8237) evaluated in the 13 NHL cell lines phenocopies shRNA knockdown with associated inhibition of proliferation (IC50=0.05 mM) and promotes apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Combination of MLN8237 with a microtubule targeting agent (docetaxel) to abrogate the spindle checkpoint is synergistic in a sequence specific manner. Moreover, MLN8237 effectively inhibits Aurora A auto-phosphorylation and eliminates phospho-histone H3 (Ser10) phosphorylation. Currently, 2 mouse MCL (Granta 519) xenograft models are underway evaluating tumor growth inhibition (TGI), safety and survival of MLN8237 alone and in combination with docetaxel or rituximab respectively. Preliminary data show that MLN8237 is synergistic with docetaxel in TGI in the MCL xenograft model. Together the data suggest inhibition of Aurora kinases may offer a promising treatment strategy for patients with aggressive B-cell NHL [Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.]. Disclosures: Rimsza: High Throughput Genomics:.

Carfilzomib (CFZ): A Novel Proteasome Inhibitor That Induces Cell Cycle Arrest and Cell Death In Rituximab-Chemotherapy Resistant Lymphoma and Potentiates the Anti-Tumor Activity of Chemotherapeutic Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4908-4908
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Gregory P. Kaufman ◽  
Cory Mavis ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Cycle Arrest ◽  
Anti Tumor Activity

Abstract Abstract 4908 Rituximab-chemotherapy relapsed/refractory B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. Targeting the ubiquitin-proteasome system using bortezomib (BTZ) has resulted in significant anti-tumor activity and potentiates the effects of chemotherapy/biologic agents in multiple myeloma, and to a lesser degree, B-cell lymphoma. CFZ is as a novel proteasome inhibitor which is selective and structurally distinct from BTZ. In an attempt to characterize the biological activity of CFZ, we evaluated its anti-tumor activity in several lymphoma pre-clinical models. Rituximab-chemotherapy sensitive cell lines (RSCL), rituximab-chemotherapy resistant cell lines (RRCL), as well as primary tumor cells derived from patients with de novo or relapsed/refractory B-cell lymphoma, were exposed to escalating doses of CFZ or BTZ (1-7.5nM) alone or in combination with doxorubicin, paclitaxel, or gemcitabine for 24, 48 and 72hours. Cell viability was determined by cell titer glow luminescent assay and cell cycle was analyzed by FASCan DNA methodology. Patient-derived lymphoma cells were isolated from fresh biopsy tissue via negative selection using magnetic beads. Western blots were performed using cell lysates from CFZ, BTZ or control-treated cells to detect PARP-cleavage and/or changes in Bcl-2 family members. CFZ was more active than BTZ and exhibited dose-dependent and time-dependent cytotoxicity against RSCL, RRCL, and primary tumor cells. We found a 10-fold concentration difference between CFZ and BTZ activity. In vitro exposure of RRCL or RSCL to CFZ resulted in G2/M phase cell cycle arrest. In addition, CFZ exposure resulted in the up-regulation of Bak and Noxa levels and subsequent PARP cleavage in RRCL. Finally, CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumor activity of paclitaxel and gemcitabine in B-cell lymphoma cell lines. In summary, our data strongly suggest that CFZ is a novel and potent proteasome inhibitor which is able to: overcome resistance to some conventional chemotherapeutic agents, upregulate proapoptotic proteins to enhance cell death, and induce G2/M cell cycle arrest in lymphoma cells. Our preclinical data supports future clinical evaluation of CFZ in patients with refractory B-cell lymphoma. (Supported by USPHS grant R01 CA136907-01A1 from the National Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of Nuclear Factor Kappa B (NFkB)/Interferon Regulatory Factor 4 (IRF4) Signaling Pathway in Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) By Combining MLN4924, a Novel NEDD8 Activating Enzyme Inhibitor (NAE) and Ibrutinib

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1768-1768 ◽  
Author(s):  
Pallawi Torka ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Sarah Belliotti ◽  
Cory Mavis ◽  
Juan Gu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Clinical Studies ◽  
Therapeutic Strategy ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Chemotherapy Agents

Abstract Identification of critical signaling pathways required for the development, maintenance and progression of specific subtypes of DLBCL is essential in order to develop novel therapeutic agents, especially for patients with relapsed/refractory disease or those not eligible for high-dose chemotherapy and autologous stem cell support (HDC-ASCT). Gene expression profiling (GEP) studies have identified three distinct subtypes of DLBCL- ABC-DLBCL, germinal center B-cell (GCB) DLBCL and primary mediastinal B-cell lymphoma (PMBL). Pre-clinical and clinical studies suggest that ABC-DLBCL is driven by an abnormally high NFκB activity that deregulates expression of Bcl-2 family proteins, and is associated with resistance to chemotherapy agents resulting in inferior clinical outcomes when compared to GCB-DLBCL or PMBL. In ABC-DLBCL, activation and maintenance of NFκB is mediated by the B-cell receptor (BCR) signaling pathway, the ubiquitin-proteasome system (UPS), oncogenic CARD11 or MYD88 mutations, and/or the effect of IRF4/SPIB on CARD11 or MYD88. Optimal NFκB targeting is an attractive therapeutic strategy in ABC-DLBCL and ongoing clinical trials are incorporating specific inhibitors (bortezomib, lenalidomide, or ibrutinib) in combination with chemo-immunotherapy in the front-line setting. On the other hand, multi-step targeting of the NFκB signaling pathway in ABC-DLBCL using investigational and currently available small molecule inhibitors could result in novel, active, and potentially less toxic regimens for ABC-DLBCL patients. To this end we studied the biological activity of MLN4924, a NAE inhibitor that selectively blocks the UPS up-stream by preventing activation of a subset of ubiquitin ligases known as cullin-RING ligases in combination with ibrutinib in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing ABC- and GCB-DLBCL were exposed to MLN4924. Changes in cell viability, cell cycle/NFκB activity, and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. Subsequently, ABC- or GCB-DLBCL cell lines and tumor cells isolated from previously untreated or relapsed/refractory B-cell lymphoma were exposed to MLN4924 in combination with various chemotherapy agents or other available NFkB inhibitors (i.e. ibrutinib) for 24 or 48 hrs. Changes in viability were determined and coefficient of synergy was calculated using the CalcuSyn software. MLN4924 induced cell death in ABC-DLBCL cell lines and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR),due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. MLN4924 enhanced activity of cytarabine, cisplatin, doxorubicin and etoposide in ABC-, but not in the GCB-DLBCL cell lines. MLN4924 also exhibited synergistic effects when combined with ibrutinib in ABC-DLBCL cell lines at the doses tested. Our data suggests that multi-step targeting of NFκB signaling pathway in ABC-DLBCL is a viable therapeutic strategy and supports further in vivo pre-clinical studies and clinical studies in relapsed/refractory or HDC-ASCT ineligible ABC-DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1366-1366 ◽  
Author(s):  
Miki Kobayashi ◽  
Satoki Nakamura ◽  
Takaaki Ono ◽  
Yuya Sugimoto ◽  
Naohi Sahara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Aurora A ◽  
Aurora Kinases

Abstract Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells. Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively. Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot. Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.

The Combination of Romidepsin and Bortezomib Results in Synergistic Induction of Apoptosis in Human B-Lymphoma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1689-1689 ◽  
Author(s):  
Deshpande S. Deshpande ◽  
Mary Jo Lechowicz ◽  
Rajni Sinha ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Mtt Assay ◽  
Research Funding ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Sequence Specificity ◽  
B Cell Malignancies

Abstract Abstract 1689 Poster Board I-715 Introduction The use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental Design Daudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. Results The combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of < 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. Conclusion The combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. Disclosures Lechowicz: Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

Combined Inhibition of Janus and Aurora Kinases by the Novel Small Molecule Inhibitor AZD1480 Induces Cell Death and Downregulates PD-L1 and PD-L2 Expression In Hodgkin Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2848-2848
Author(s):  
Enrico Derenzini ◽  
Daniela Buglio ◽  
Hiroshi Katayama ◽  
Yuan Ji ◽  
Subrata Sen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Death ◽  
Cell Lines ◽  
Immune Escape ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Aurora Kinases ◽  
Mitotic Block ◽  
Dose Dependent

Abstract Abstract 2848 Hodgkin Lymphoma (HL) cell proliferation and survival is sustained by a complex network of cytokine signaling, involving the Hodgkin and Reed-Sternberg cells and tumor microenvironment. Following cytokine stimulation, JAK-STAT activation promotes the transcription of target genes involved in proliferation, survival, and immune escape. Programmed Death-ligands 1 and 2 (PD-L1 and PD-L2) and the Th2 chemokine TARC are immune-modulators involved in immune evasion, respectively through inhibition of effector T cell function (PD-L1, PD-L2) and attraction and homing of Th2 cells (TARC). Aurora kinases are frequently overexpressed in human cancers and play essential functions in chromosome alignment and cytokinesis. The role of Aurora kinases in Hodgkin lymphomagenesis is not defined yet. In this study we report the activity profile of the JAK2 inhibitor AZD1480 in HL cell lines (HD-LM2, L-428, KM-H2, L-540). To assess the effect of AZD1480 on cell proliferation, cells were incubated with increasing concentrations of AZD1480 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs). A significant growth inhibition was evident after 72 hrs of incubation, specially using the high doses of AZD1480 (5μM). The L-540 cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with AZD1480 1μM. Inhibition of STAT3, STAT5 and STAT6 phosphorylation in the L-540, L-428 and HD-LM2 cell lines was observed with concentrations equal to 0.1 μM or higher. Using Annexin V- propidium iodide staining, we found that AZD1480 induced cell death by apoptosis in a dose dependent manner after 72 hrs of incubation when a high concentration (5μM) of the drug was used. Lower concentrations of AZD1480 (1μM) promoted a statistically significant increase in cell death only in the L-540 and to a lesser extent in the L-428 cell line. Consistent with this data, also caspase 9, 3 and PARP cleavage was observed in all the cell lines exposed to AZD1480 5 μM. AZD1480 5μM promoted a marked increase in the G2/M fraction in all the cell lines as soon as 24 hrs after incubation, especially in the HD-LM2 and L-428 cell lines. Treatment with lower doses (1μM) did not affect significantly the cell cycle. Since AZD1480 was also reported to inhibit Aurora A kinase at nanomolar concentrations in enzymatic assays, we assessed if the significant increase in the G2/M fraction was related to the inhibition of the Aurora A kinase. We evaluated the levels of autophosphorylation on Thr-288 by western blotting. Cells were pretreated with Nocodazole 400 ng/ml for 18 hrs in order to achieve a mitotic block, and then exposed to AZD1480 (1-5μM) and/or the proteasome inhibitor MG132 (20μM) (in order to prevent the potential overriding of the Nocodazole induced mitotic block), for 3 hours. A dose-dependent inhibition of Aurora A was detected in all the cell lines, with a complete abrogation when higher doses of AZD1480 were used (5μM). These findings are consistent with the analysis of the cell cycle fractions, showing dose-dependent changes of the cell cycle at 24 hrs following incubation with AZD1480. AZD1480 also decreased the secretion of key cytokines involved autocrine and paracrine survival loops and immune escape. Following incubation with AZD1480 1μM for 72 hrs cell culture supernatants were analyzed by ELISA: decreased levels of IL-6, IL-13, TARC, and IL-21 were observed in HD-LM2, L-428 and L-540 cells. Moreover we assessed the expression of PD-L1 and PD-L2 by flow cytometry and observed significant downregulation in the PD-L1/PD-L2 overexpressing cell lines (L-540 and HD-LM2). These data suggest that AZD1480 has a pleiotropic mechanism of action in HL by targeting the JAK-STAT and the Aurora kinase pathway, and by altering the pattern of cytokine and chemokine secretion and the expression of factors involved in immune escape. Our study provides the rationale for further clinical investigation of AZD1480 in HL. Disclosures: No relevant conflicts of interest to declare.

The Bioactive Polyphenol Curcumin (diferuloylmethane) In Human Apolipoprotein A-1 Nanodisks Enhances Apoptosis and G1 Cell Cycle Arrest In Mantle Cell Lymphoma Compared with Free Curcumin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3934-3934
Author(s):  
Amareshwar T.K. Singh ◽  
Mistuni Ghosh ◽  
C. Shad Thaxton ◽  
Trudy M. Forte ◽  
Robert O. Ryan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 3934 Background: Mantle cell lymphoma (MCL) is a pre–germinal center neoplasm characterized by cyclin D1 overexpression resulting from translocation of the cyclin D1 gene on 11q13 to the promoter of the immunoglobulin heavy chain locus on 14q32. Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. The bioactive polyphenol curcumin (Curc), derived from the rhizome of Curcuma longa Linn, has been shown to have pleiotropic activities related to its complex chemistry and its influence on multiple signaling pathways including NF-kB, Akt, Nrf2 and pathways involved in metastasis and angiogenesis. Curc has been shown to cause growth arrest and apoptosis of BKS-2 immature B-cell lymphoma by downregulating growth and survival promoting genes (Clin Immunol 1999; 93:152). However, because of poor aqueous solubility Curc has had limited clinical utility, so investigators have explored nanoparticle drug delivery approaches (J Nanobiotech 2007, 5:3, MCT 2010; 9:2255). We reasoned that effective and targeted drug delivery by nanoparticles required appropriate receptors to facilitate binding. We therefore screened lymphoma cell lines for receptors that recognize apolipoprotein (apo) A-1. We hypothesized that a novel discoidal nanoparticle (ND) consisting of apoA-1, phospholipid and Curc (Curc ND) would bind to such receptors to facilitate drug delivery. Methods: We compared biologic activity of free Curc vs. Curc-ND in MCL cell lines expressing receptors for apoA-1. Cell lines were grown and maintained in culture, treated, and apoptosis and cell cycle progression was measured by flow cytometry. Relevant signaling intermediates and presence of apoA-1 receptors were measured by immunoblotting using specific antibodies. Results: Granta and Jeko cells (both MCL cell lines) expressed apoA-1 receptors including class B scavenger receptor (SR-B1) and the ATP-binding cassette transporter of the sub-family G1 (ABCG1). To compare the pro-apoptotic effect of free Curc and Curc-ND, Granta cells were incubated with free Curc, Curc-ND, empty ND, and medium alone (untreated). Compared to medium alone, empty ND had no effect while free Curc (20 μM) induced apoptosis. Curc-ND produced a dose-dependent increase in apoptosis, with ∼70% apoptosis at 20 μM. To investigate the mechanism of Curc-ND induced apoptosis, apoptosis-related proteins were studied in cultured Granta cells. A time-dependent decrease in caspase-9 levels was observed following incubation with Curc-ND or free Curc. The decrease in caspase-9 seen with Curc-ND, however, occurs much earlier (between 2–4 h of incubation) than for free-Curc. Caspase-3 was undetectable after 16 h with either treatment. Loss of this band implies activation of caspase-3, which was confirmed by PARP cleavage, wherein a decrease in the 116 kD band was accompanied by an increase in the 85 kD cleavage product. Unlike free Curc, Curc-ND induced PARP cleavage even at 16 h of incubation, suggesting sustained drug release. Curc-ND downregulated cyclin D1, decreased Akt phosphorylation and enhanced cleavage of caspases-9 and -3, and PARP. In addition, Curc-ND induced G1 cell cycle arrest to a greater extent than free Curc in Granta and Jeko cells (Granta: Control 34% G1, Curc 37% G1, Curc-ND 46% G1; Jeko: Control 39% G1, Curc 49% G1, Curc-ND 54% G1). Conclusion: We have shown that the MCL cell lines Granta and Jeko express apoA-1 receptors, making them likely targets for discoidal nanoscale delivery vehicles stabilized with Apo-A1. These nanodisks, when carrying the polyphenol Curc, can result in increased caspase -dependent apoptosis, cell cycle arrest, downregulation of cyclin-D1 and decreased p-Akt. These data suggest that the pleiotropic polyphenol Curc has cell killing/arrest activity in MCL and that Curc-ND may be a potential therapeutic with drug targeting ability. Disclosures: Forte: Lypro Biosciences: Employment.

The Brd-Inhibitor OTX015 Is Active in Pre-Clinical Models of Mature B-Cell Lymphoid Tumors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1657-1657 ◽  
Author(s):  
Paola Bonetti ◽  
Michela Boi ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stahis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Rt Pcr ◽  
Down Regulation ◽  
Myc Gene ◽  
Dose Dependent

Abstract Abstract 1657 Background: Bromodomain-containing proteins play an important role in gene expression regulation, via chromatin structure remodelling. Antitumor activity has been reported in acute and chronic hematological malignancies using inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a large panel of cell lines derived from mature B-cell lymphoid tumors. Material and Methods: Established human cell lines derived from 13 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphoma (MCL), three splenic marginal zone lymphoma (SMZL) and from three multiple myeloma (MM) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72 hours exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: OTX015 demonstrated anti-proliferative activity in DLBCL cell lines (median IC50 0.192μM; range 0.069–12.68μM). Similar results were obtained on SMZL (median IC50 0.165μM, range 0.105–0.24μM), and on MM cell lines (median IC50 0.449μM; range 0.06–0.7μM). Conversely, MCL cell lines appeared less sensitive to OTX015 (median IC50 2.01μM; range 1.22- >15μM). Among DLBCL cell lines, there was no significant difference based upon the cell of origin of the cell lines. OTX105 caused a cell cycle arrest in G1 in a dose-dependent manner in 5/5 DLBCL and 3/3 MM cell lines, without an increase in cell death. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in 1/1 sensitive DLBCL cell line. In order to understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after 24h treatment with increasing doses. We observed a dose-dependent suppression of MYC mRNA by OTX015 in 4/5 DLBCL and in 2/2 MM cell lines. In DLBCL, down-regulation of MYC mRNA was observed within 1h after treatment with OTX015, suggesting a direct effect of the compound on the MYC gene. To determine whether the suppression of MYC gene by OTX015 was reversible, DLBCL cell lines were treated for 2h with OTX015 and then the inhibitor was removed from the media. MYC mRNA suppression appeared reversible, as shown in DLBCL cell lines, which, after 2h exposure to OTX015, showed a time-dependent restoration of MYC mRNA expression to untreated levels after 2–3h. In one of the most sensitive DLBCL cell lines no MYC mRNA down-regulation was observed after treatment, suggesting that alternative pathways can be affected by BRD-inhibition. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several cell lines representative of mature B-cell tumors. An apparently reversible down-regulation of MYC mRNA was commonly observed, appearing as a possible mechanism of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in mature B-cell origin malignancies. A phase I trial is scheduled to start in 2012. Disclosures: Bonetti: OncoEthix SA: Research Funding. Inghirami:OncoEthix SA: Research Funding. Noel:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Bertoni:OncoEthix SA: Research Funding.

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 Combination Benefit of Capivasertib and Venetoclax in Preclinical Models of Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1870-1870
Author(s):  
Brandon Willis ◽  
India Neveras ◽  
Hannah Dry ◽  
Wendan Xu ◽  
Yang Li ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Mouse Models ◽  
Cell Lines ◽  
Stock Options ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Growth Inhibition ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy among adults and despite approximately 65% of patients with DLBCL being cured with RCHOP therapy, nonresponsive and relapsed patients have inadequate treatment options, highlighting the importance for innovative treatment regimens. Blockade of B-cell receptor (BCR) downstream signaling components with various targeted agents is emerging as a clinically tractable treatment strategy across multiple B-cell malignancies. Protein Kinase B (AKT) signaling downstream of the BCR complex has been shown to be a central node in germinal center B-cell (GCB) DLBCL and the potent, selective inhibitor of AKT1, AKT2, AKT3, capivasertib, currently being evaluated in multiple clinical trials by targeting AKT-driven solid cancers, has been shown to induce apoptosis in a subset of GCB-DLBCL cell lines and cause tumor stasis in xenograft mouse models (Erdman et al., 2017). Since the monotherapy capivasertib responses in GCB DLBCL models are partial and lack durability, we hypothesized a combination approach could deliver even greater therapeutic benefit. To identify optimal partners, we conducted a capivasertib centric in vitro combination screen with specific with BH3 family members across a panel of 15 DLBCL cell lines, which revealed a synergistically active combination with the BCL2 inhibitor, venetoclax which is currently being evaluated in DLBCL. The activity was specifically enhanced in cell lines of the GCB subtype, with 4 PTEN del and 2 PTEN wt cell line models showing combination benefit. To determine the ability of this combination to drive stronger and durable responses, we assessed capivasertib and ventoclax activity in xenograft mouse models using two GCB-DLBCL cell line lines, SUDHL4 (PTEN wt) and WSU-DLCL2 (PTEN del). Oral administration of either monotherapy capivasertib (130 mg/kg BID, 4-day on/3-day off) or venetoclax (100 mg/kg QD) provided partial tumor growth inhibition (capivasertib TGI = 74% in SUDHL4 and 29% in WSU-DLCL2, and venetoclax TGI = 46% in SUDHL4 and 0% in WSU-DLCL2), whereas the combination of capivasertib and venetoclax both on a 4-day on/3-day off schedule produced complete tumor regression (100% regression) in both xenograft GCB cell line models during the dosing period. Notably, in both xenograft models all mice (5/5 per model) remained tumor free for at least 30 days following dosing cessation demonstrating high durability of response for the combination. Additionally, this combination is currently being evaluated in clinically relevant GCB and non-GCB PDX mouse models. Taken together, our results provide preclinical evidence for the rational combination of AKT and BCL-2 blockade with capivasertib and venetoclax respectively in patients with relapsed/refractory GCB-DLBCL. Disclosures Willis: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Neveras: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Dry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mongeon: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Rosen: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mettetal: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Barry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options.

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