scholarly journals The Combinatorial Activity of Eftozanermin (ABBV-621), a Novel and Potent TRAIL Receptor Agonist Fusion Protein, in Pre-Clinical Models of Hematologic Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-41
Author(s):  
Morey L Smith ◽  
Sha Jin ◽  
Dong Chen ◽  
Haichao Zhang ◽  
Jason Huska ◽  
...  
Keyword(s):  
Cell Death ◽  
Fusion Protein ◽  
Cell Line ◽  
Receptor Agonist ◽  
B Cell Lymphoma ◽  
Synergistic Activity ◽  
Trail Receptor ◽  
Phase I Clinical Trials ◽  
Single Chain ◽  
Publicly Traded

Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209). To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Thein vitroresults led to selection of agents to combine with ABBV-621 forin vivostudies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Smith: AbbVie:Current Employment, Current equity holder in publicly-traded company.Jin:AbbVie:Current Employment, Current equity holder in publicly-traded company.Chen:AbbVie:Current Employment, Current equity holder in publicly-traded company.Zhang:AbbVie:Current Employment, Current equity holder in publicly-traded company.Huska:AbbVie:Current Employment, Current equity holder in publicly-traded company.Widomski:AbbVie:Current Employment, Current equity holder in publicly-traded company.Bontcheva:AbbVie:Current Employment, Current equity holder in publicly-traded company.Buchanan:AbbVie:Current Employment, Current equity holder in publicly-traded company.Morgan-Lappe:AbbVie:Current Employment, Current equity holder in publicly-traded company.Phillips:AbbVie:Current Employment, Current equity holder in publicly-traded company.Tahir:AbbVie:Current Employment, Current equity holder in publicly-traded company.

scholarly journals Re-Wiring BCL-2 Family Anti-Apoptotic Protein Dependencies through Modulating Phosphorylation to Re-Sensitize Venetoclax-Resistant Lymphoid Malignancies to BCL-2 Inhibition

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Stephen Jun Fei Chong ◽  
Mary C Collins ◽  
Liam Hackett ◽  
Matthew S. Davids
Keyword(s):  
Cell Death ◽  
Protein Expression ◽  
Cell Line ◽  
Research Funding ◽  
Family Members ◽  
B Cell Lymphoma ◽  
Prolonged Treatment ◽  
Lymphoid Malignancies ◽  
Phosphatase Inhibitor ◽  
Apoptotic Protein

Introduction Resistance to apoptosis is a hallmark of cancer, and modulation of BCL-2 family proteins is an important mediator of such resistance in hematologic malignancies. Despite the clinical efficacy of the BCL-2 inhibitor venetoclax (VEN), prolonged treatment may lead to resistance, such as the BCL2 G101V mutation (Blombery et al, Blood, 2020); however, over half of VEN resistant cases are not explained by known genetic mechanisms. Phosphorylation of BCL-2 at serine-70 (S70pBCL2) or of MCL-1 at threonine-163 (T163pMCL1) have been shown to increase sequestration of the pro-apoptotic protein BAX (Deng et al, JNCI, 2000) and stabilize the level of anti-apoptotic protein MCL-1 (Wang et al, Mol Cancer, 2014), respectively. We hypothesized that the increase in post-translational modifications of BCL-2 family members, in particular S70pBCL2 and T163pMCL1, are novel mechanisms of functional VEN resistance in lymphoid malignancies. We further hypothesized that the FDA-approved phosphatase activator drug FTY720 (fingolimod) would de-phosphorylate these BCL-2 family members and thereby re-sensitize malignant lymphoid cells to VEN-induced apoptosis. Methods A VEN resistant diffuse large B-cell lymphoma cell line (OCI-Ly1-R) as well as peripheral blood mononuclear cells from 12 previously untreated CLL patients co-cultured with human stromal NK-Tert cells were treated ex vivo with VEN +/- FTY720. A VEN sensitive cell line (OCI-Ly1-S) was treated with VEN +/- a phosphatase inhibitor okadaic acid (OA). Western blot was used to evaluate changes in S70pBCL2 and T163pMCL1 protein levels. BH3 profiling via flow cytometry was performed to determine the survival dependence on anti-apoptotic BCL-2 family members via cytochrome c release in response to specific BH3-only peptides and drugs such as VEN applied directly to mitochondria (Ryan et al, Biol Chem, 2016). Cell viability assays (CellTiter-Glo, Trypan Blue and Annexin/Hoechst) were employed to investigate the effects of FTY720 on OCI-Ly1-R resistance to VEN. The BCL-2-BAX interaction was investigated using co-immunoprecipitation in VEN resistant and sensitive cells following treatment with VEN +/- FTY720. T-test, ANOVA and multiple comparison with a statistical significance set at 2-tailed p ≤ 0.05 were used. Results OCI-Ly1-R cells displayed higher S70pBCL2, T163pMCL1 and MCL-1 expression compared to OCI-Ly1-S cells. Notably, the increase in S70pBCL2 was associated with reduced response of VEN-mediated BCL-2-BAX dissociation, while the increase in T163pMCL1 was accompanied by enhanced MCL-1 protein expression. Using BH3 profiling, we found that the increase in S70pBCL2, T163pMCL1 and MCL-1 expression were functionally associated with a decrease in BCL-2 survival dependence (-79.1%, 1μM VEN, P < 0.0001) and an increase in MCL-1 dependence (+52%, 10μM MS1, P < 0.0001) (Fig. A). The addition of FTY720 reversed these observations in OCI-Ly1-R cells, where we observed a decrease in S70pBCL2, T163pMCL1 and MCL-1 protein expression, BCL-2 and BAX interaction, as well as a "re-wired" functional dependence toward BCL-2 (-21.6% 10μM MS1, +27.9% 1μM VEN, P < 0.0001) (Fig. B). Importantly, pre-treatment with FTY720 re-sensitized OCI-Ly1-R cells to VEN-induced cell death (+56.1%, P = 0.0001) (Fig. C). Conversely, treatment with a phosphatase inhibitor, OA, led to an increase in S70pBCL2, T163pMCL1 and MCL-1 expression as well as reduced late death of OCI-Ly1-S cells (-60%, P = 0.0018). We validated our cell line results in primary CLL cells, and again the combination of FTY720 and VEN similarly reduced S70pBCL2, T163pMCL1 and MCL-1 expression, increased BCL-2 dependence, and enhanced VEN-induced cell death (+23.6%, P < 0.0001). Conclusion Increased S70pBCL2 and T163pMCL1 are associated with VEN resistance, in part by inhibiting VEN-induced BCL-2-BAX dissociation and switching the functional survival dependence from BCL-2 to MCL-1. FTY720 re-sensitizes VEN resistant cells by reducing S70pBCL2, T163pMCL1 and MCL-1 expression, dissociating BAX from BCL-2 and "re-wiring" the survival dependence to BCL-2. These preclinical findings support the exploration of this strategy clinically in patients with VEN resistant lymphoid malignancies. Disclosures Davids: Sunesis: Consultancy; AbbVie: Consultancy; Surface Oncology: Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Zentalis: Consultancy; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Merck: Consultancy; Research to Practice: Honoraria.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

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

Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3288-3288
Author(s):  
Brian Gaudette ◽  
Neal N Iwakoshi ◽  
Lawrence H. Boise
Keyword(s):  
Cell Death ◽  
Cell Differentiation ◽  
B Cells ◽  
Er Stress ◽  
B Cell ◽  
Cell Line ◽  
Plasma Cell ◽  
B Cell Lymphoma ◽  
Plasma Cell Differentiation ◽  
Dependent State

Abstract Abstract 3288 Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1142-1142
Author(s):  
Jennifer Totman ◽  
Dorothy Brach ◽  
Vinny Motwani ◽  
Selene Howe ◽  
Emily Deutschman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Publicly Traded ◽  
The Past ◽  
Molecule Inhibitor

Abstract Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types. Methods: Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays. Results: Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to >10 μM. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated > 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting > 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. In vitro synergistic antiproliferative activity was also observed when EZM0414 was combined with certain SOC agents for MM and DLBCL. Conclusions: Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL. Disclosures Totman: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Brach: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Motwani: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Howe: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Deutschman: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Lampe: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Riera: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Eckley: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Alford: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Duncan: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Farrow: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Dransfield: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Raimondi: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Thomeius: Foghorn Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cosmopoulos: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Kutok: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company.

JQ1, a Potent c-MYC Inhibitor Overcomes Rituximab-Chemotherapy Resistance in Lymphoma Pre-Clinical Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2761-2761
Author(s):  
Juan J Gu ◽  
Qunling Zhang ◽  
Cory Mavis ◽  
Myron S. Czuczman ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Board ◽  
Synergistic Activity ◽  
Targeted Agents ◽  
Chemotherapy Agents

Abstract Background: The poor clinical outcomes of patients with aggressive B-cell lymphoma in the post-rituximab era, stress the need to identify and/or optimize novel targeted agents. Several retrospective and prospective clinical studies had demonstrated that C-myc expression correlates with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory diffuse large B-cell lymphoma (DLBCL). To this end, we evaluated the therapeutic effects of targeting C-myc using JQ1, a novel bromodomain inhibitor in rituximab-sensitive or -resistant models. Methods: A panel of rituximab-sensitive (RSCL) or rituximab-resistant (RSCL) cell lines was exposed to JQ1 (0-100 µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently lymphoma cells were exposed to JQ1 or vehicle and various chemotherapy agents such as doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), Ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Results: In vitro exposure of RRCL and to a lesser degree RSCL to JQ1 resulted in a dose- and time-dependent cell death. Strong synergistic activity was observed when JQ1 was combined with doxorubicin, dexamethasone bortezomib or carfilzomib in vitro. Cell cycle analysis demonstrated that in vitro of RSCL or RRCL to JQ1 resulted in G1 cell cycle arrest. Conclusions: In summary, our data suggests that targeting C-myc expression using JQ1 results in anti-tumor activity against RSCL and RRCL. In addition, JQ1 exhibited synergistic activity when combined with chemotherapy agents (doxorubicin or dexamethasone) or targeted agents (bortezomib or carfilzomib). On going studies are aimed to study the mechanisms by which c-myc inhibition results in cell death in RSCL and RRCL. JQ1 is a distinct targeted agent undergoing clinical evaluation in patients with relapsed/refractory lymphomas. Molecular studies dissecting the cellular pathways affected by JQ1 are important in order to further advance the clinical development of c-myc inhibitors in lymphoid malignancies. (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 Czuczman: Boehringer-Ingelheim: Other: Advisory Board; Immunogen: Other: Advisory board; MorphoSys: Consultancy; Celgene: Employment.

Targeted cytokine delivery to neuroblastoma

2002 ◽  
Vol 30 (4) ◽  
pp. 518-520 ◽  
Author(s):  
P. K. Dehal ◽  
M. J. Embleton ◽  
J. T. Kemshead ◽  
R. E. Hawkins
Keyword(s):  
Fusion Protein ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Lymphoid Cells ◽  
Human Neuroblastoma Cell ◽  
Single Chain ◽  
Human Neuroblastoma ◽  
Gm Csf ◽  
Single Chain Fv

The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.

Obatoclax and Bortezomib Therapy Results in Disruption of the p53-Mediated Apoptosis/Autophagy Pathway and Is Associated with Potent Synergistic Anti-Tumor Activity against Rituximab-Chemotherapy-Sensitive and -Resistant B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 288-288 ◽  
Author(s):  
Francisco Hernandez-Ilizaliturri ◽  
Ping-Chiao Tsio ◽  
Ryan Campagna ◽  
Cory Marvis ◽  
Wasif Riaz ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Synergistic Activity ◽  
Knock Down ◽  
Anti Tumor Activity

Abstract Abstract 288 Interaction between the members of the BH3 domain family of proteins plays an important role in the development, progression, and prognosis of various subtypes of B-cell lymphoma. Therapies that selectively induce a pro-apoptotic environment are an attractive strategy to overcome chemotherapy-resistance in B-cell lymphoma. The proteasome is an important regulator of various members of Bcl-2 family proteins. We previously demonstrated that obatoclax, a novel BH3 mimetic, was able to enhance the anti-tumor activity of rituximab and chemotherapy agents and induced both apoptosis and autophagy in B-cell lymphoma. In an attempt to increase the therapeutic options for B-cell lymphoma patients we studied the biological effects of obatoclax in combination with bortezomib in a panel of rituximab-[chemotherapy]-sensitive (RSCL) and rituximab-[chemotherapy]-resistant cell lines (RRCL), as well as primary tumor cells isolated from 45 NHL patient biopsy samples with various subtypes of B-cell lymphoma: (ie including, DLBCL, follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and Hodgkin lymphoma (HL)]. Patient-derived primary tumor cells were isolated from fresh biopsies by negative selection using magnetic beads. Transient knock-down of p53 and Noxa were performed to determine the role of p53 in the anti-tumor activity of bortezomib or obatoclax, respectively. NHL cells were exposed in vitro to escalating doses of obataclox (0, 2, 5, 10 and 20mM) and/or bortezomib (0, 2, 10 and 20nM) for 24 and 48 hrs. Cell death was determined by the cell glow luminescent assay and DNA synthesis was evaluated by standard [3H]-Thymidine incorporation assays at 24 and 48 hrs. Changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. In vitro exposure of RRCL, RSCL, and primary tumor cells to the combination of obatoclax plus bortezomib demonstrated significant synergistic activity compared to controls. Patients with DLBCL (n=15) and FL (N=12) demonstrated significant sensitivity to this combination. Of note, activity was observed in patients with either de novo or relapsed/refractory germinal B-cell (GBC) or activated B-cell (ABC) DLBCL (as characterized by the Han's criteria). Additionally, cell death induced by obatoclax or bortezomib could be inhibited by transient knock-down of p53 or Noxa, respectively. In summary, deregulation of apoptosis by BH3 inhibition with obatoclax and bortezomib results in cell death and antiproliferation not only in RSCL and RRCL, but also in primary tumor cells derived from “treatment-naïve or refractory” DLBCL and FL patients. Data strongly suggests that both p53 and Noxa have pivotal roles in response to obatoclax and bortezomib. The combination of obatoclax plus bortezomib has the potential of becoming a novel and potent therapeutic strategy in the treatment of B-cell lymphoma in the future. Research, in part, supported as part of a subproject on NIH PO1 grant CA103985-1 awarded to the Garden State Cancer Center, Belleville, NJ and NHI R-01 grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute Disclosures: No relevant conflicts of interest to declare.

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