Z-Guggulsterone Downregulates Survivin and Induces Cell Death in Large B Cell Lymphoma Cells In Vitro.

Abstract Introduction: Survivin is a member of the Inhibitor of Apoptosis Proteins and has recently gained attention as a possible therapeutic target in malignancies, due to its dual role both as an antiapoptotic protein and as a cell cycle regulator. It is overexpressed in malignant cells and confers resistance to chemotherapy and other stimuli triggering apoptosis. Z-Guggulsterone (Z-GGS) is a plant sterol, which has been used in inflammatory conditions and has been recognized as a potent NF-kB suppressor. Since Survivin, as well as other antiapoptotic proteins, are under NFkB regulation, we studied the effect of Z-GGS on two B-cell lymphoma cell lines. Methods: DB and HT cell lines were treated with increasing concentrations (10μM, 20μM and 30μM) of Z-GGS, for 24, 48 and 72 hours. Survivin expression was tested with Flow Cytometry and Survivin transcripts were measured with quantitative real time PCR using the Universal Probe Library hydrolysis probes and expressed as Survivin/abl ratio. Cell viability was assessed with the MTT assay. Results: Both cell lines were positive for Survivin at baseline by flow cytometry (66% of total cells for DB and 95% for HT). Treatment of DB cells with 10, 20 and 30μM Z-GGS resulted in a 44%, 49% and 68% reduction of Survivin expression at 24 hours, respectively, whereas the effect on HT was less prominent with a 10% reduction at 24 hours with 30μM Z-GGS. Survivin transcripts decreased as well, with the maximum effect observed at 72 hours with 30μM Z-GGS for both cell lines: Survivin/abl was 0.009 for untreated cells vs 0.0008 with 30μM Z-GGS for DB cells and 0.0135 vs 0.0005 for HT cells. Linearity was observed for increasing concentrations of Z-GGS at 72 hours. Cell viability was practically unaffected at any time point with 10 and 20μM Z-GGS for both cell lines, whereas 30 μM Z-GGS resulted in a 63% and 78% cell death at 48 and 72 hours respectively for DB cells and 67% and 83% for HT cells. Conclusions: The steroid Z-GGS downregulates Survivin expression in B-lymphoma cells in vitro and induces cell death at 30μM concentration. Further experiments will clarify its possible role in the treatment of B-cell malignancies.

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MLN2238, a Novel and Potent Proteasome Inhibitor, Induces Caspase-Dependent Cell Death, Cell-Cycle Arrest, and Potentiates the Anti-Tumor Activity of Chemotherapy Agents In Rituximab-Chemotherapy Sensitive or Resistant B-Cell Lymphoma Cell Lines

Blood ◽

10.1182/blood.v116.21.3939.3939 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3939-3939

Author(s):

Juan Gu ◽

Patil Ritesh ◽

Cory Mavis ◽

George Deeb ◽

John Gibbs ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Anti Tumor Activity

Abstract Abstract 3939 The use of proteasome inhibitors such as bortezomib (BTZ) has generated much excitement as a potential therapeutic approach capable of effectively treating resistant/refractory lymphoid neoplasm. Clinical outcomes in multiple myeloma and relapsed mantle cell lymphoma demonstrate that these novel agents can overcome resistance demonstrated by a lack of antitumor activity to traditional salvage chemotherapeutic agents. Our group of investigators have demonstrated that proteasome inhibition using BTZ can increase pro-apoptotic Bcl-2 family member expression and restore chemotherapy sensitivity in rituximab-chemotherapy resistant cell lines (RRCL). To further develop therapeutic strategies targeting the proteasome system, we studied the anti-tumor activity and mechanisms-of-action of MLN2238, a novel irreversible proteasome inhibitor, in pre-clinical lymphoma models. Experiments were conducted in rituximab-chemotherapy sensitive cell lines (RSCL), RRCL, and in tumor cells derived from patients with de novo or relapsed/refractory B-cell lymphoma. Cells were exposed in vitro and/or ex vivo to escalating doses of MLN2238 or BTZ (0.1-10nM) +/− caspase inhibitors (zVAD-fmk or Q-VD-OPh) for 24, 48 and 72h. Differences in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay; changes in ATP content (apoptosis) were determined using the Cell Titer Glow assay. Effects on cell cycle were analyzed by the FASCan DNA method. In addition, lymphoma cells were exposed to MLN2238 or BTZ +/− doxorubicin, gemcitabine or paclitaxel and cell viability was evaluated as described above. In vitro, MLN2238 exhibited more potent concentration- and time-dependent cytotoxicity and inhibition of cell proliferation in RSCL, RRCL, as well as primary lymphoma cells than BTZ. In vitro exposure of RSCL and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL in a dose-dependent manner. Co-incubation of RSCL with bortezomib, or MLN2232 and either pan-caspase inhibitor led to a significant decrease in BTZ- or MLN2232-induced cell death. In contrast, neither zVAD-fmk nor Q-VD-OPh was capable of blocking BTZ- or MLN2232-induced cell death of RRCL. Our data suggest that BTZ and MLN2238 are also capable of inducing caspase-independent cell death in RRCL. To this regard, we found differences that RRCL are more likely to be in S phase in resting conditions when compared to RSCL. In vitro exposure of RRCL cells to MLN2232 (and to a much lesser degree BTZ) reduced RRCL S-phase and induced arrest at G2/M phase. Collectively, these data suggest that MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy sensitive or resistant cell models and potentiates the anti-tumor activity of chemotherapy agents. MLN2232 appears to posses several mechanisms-of-action (induction of apoptosis and/or cell cycle arrest) and has the potential of becoming a novel and potent target-specific therapeutic agent in the future treatment of therapy-resistant B-cell lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

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The Green Tea Extract Epigallocatechin Induces In Vitro Cell Death in Primary Human Lymphoma Cells through an ROS Dependent Mechanism.

Blood ◽

10.1182/blood.v108.11.234.234 ◽

2006 ◽

Vol 108 (11) ◽

pp. 234-234 ◽

Cited By ~ 1

Author(s):

Tait D. Shanafelt ◽

Yean K. Lee ◽

Susan M. Geyer ◽

Deanna Grote ◽

Mary Stenson ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Green Tea ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Primary Lymphoma ◽

Lymphoma Cells

Abstract BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

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Bio-Inspired Functional Lipoprotein-like Nanoparticles (Flip-NPs) Cause Cholesterol Starvation and Ferroptosis in B-Cell Lymphomas: Studies in Cell Lines, Xenograft Models and Primary Patient Samples

Blood ◽

10.1182/blood-2019-127805 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2857-2857

Author(s):

Jonathan Rink ◽

Adam Yuh Lin ◽

Shuo Yang ◽

Amir Behdad ◽

Reem Karmali ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cholesterol Reduction ◽

Xenograft Models

Introduction: Hematologic malignancies, including B cell lymphomas such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), have increased demands for cholesterol and cholesteryl esters to maintain membrane anchored pro-proliferative and pro-survival signaling pathways, including B cell receptor signaling. Recent evidence suggests that certain cancer cell lines, including several anaplastic large T cell lymphoma (ALCL) cell lines, are auxotrophic for cholesterol and are sensitive to cholesterol reduction-induced ferroptosis (Garcia-Bermudez, Nature 2019), an iron dependent form of programmed cell death characterized by accumulation of lipid peroxides. We have developed a cholesterol depleting functional lipoprotein-like nanoparticle (Flip-NP) that specifically targets the high-affinity HDL receptor, scavenger receptor type B1 (SCARB1), which maintains cellular and cell membrane cholesterol homeostasis. Our prior data demonstrated that Flip-NPs induce B cell lymphoma cell death in vitro and in in vivo xenograft models. Accordingly, we hypothesized that the mechanism of cell death by Flip-NPs in B cell lymphomas is ferroptosis, and that Flip-NPs would be potent therapy for an expanded number of cholesterol-addicted malignancies, including ALCL. Methods: After informed consent, primary B cell lymphoma cells were isolated from excisional biopsies from patients with FL or DLBCL. The SUDHL4 [germinal center (GC) DLBCL], Ramos [Burkitt's lymphoma], SUDHL1 [ALCL] and SR-786 [ALCL] cell lines were used for in vitro experiments. SCARB1 expression was quantified using flow cytometry and western blot analysis. Cell viability was quantified using the MTS assay and flow cytometry. Ferroptosis was measured using the lipophilic antioxidant ferrostatin-1 or the iron chelator deferoxamine. Gene expression changes were quantified using RT-qPCR. Lipid peroxidation was measured using C11-BODIPY and flow cytometry. SUDHL1 and SUDHL4 flank tumor xenografts were initiated in SCID-beige mice, with Flip-NPs administered 3 times per week IV. Results: Primary B cell lymphoma cells were isolated from patients with FL (n=4) or DLBCL (n=2), and all samples expressed some level of SCARB1 by flow cytometry. Flip-NPs increased cell death in 3 of the 4 FL samples and 1 of 2 DLBCL samples. In Ramos and SUDHL4 cells, RT-qPCR data showed that Flip-NP-mediated cholesterol reduction led to up-regulation of cholesterol biosynthesis genes and down-regulation of glutathione peroxidase-4 (GPX4), a critical protein responsible for degradation of lipid peroxides. Correspondingly, as shown with C11-BODIPY, Flip-NP treatment increased lipid peroxide accumulation in Ramos and SUDHL4 cells. Addition of ferrostatin-1 or deferoxamine reduced Flip-NP induced cell death, demonstrating that the mechanism-of-action of Flip-NPs involves, at least in part, ferroptosis. Given the sensitivity of cholesterol auxotrophic cell lines to cholesterol reduction-induced ferroptosis, we tested the efficacy of the Flip-NPs against cholesterol auxotrophic ALK+ ALCL cell lines SUDHL1 and SR-786. SCARB1 was expressed in both cell lines. Flip-NPs potently induced cell death in both SUDHL1 and SR-786 cells in vitro. In vivo, systemic administration of Flip-NPs reduced tumor volumes in both SUDHL4 and SUDHL1 tumor xenograft models. Conclusions: Our data show that Flip-NPs reduce GPX4 expression and increase lipid peroxide accumulation in B cell lymphoma cell lines, resulting in ferroptosis. Expanding on these results, Flip-NP efficacy was also demonstrated in cholesterol auxotrophic ALK+ ALCL cell lines and primary patient-derived B cell lymphoma cells. These in vitro results translated to in vivo murine models, as systemic administration of Flip-NPs potently reduced DLBCL and ALK+ ALCL tumor xenograft burden. Flip-NPs are a molecularly targeted, first-in-class therapy that may be effective for malignancies reliant upon cellular cholesterol. Disclosures Behdad: Pfizer: Other: Speaker; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees. Karmali:Astrazeneca: Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau. Thaxton:Zylem: Other: Co-founder of the biotech company Zylem. Gordon:Juno/Celgene: Other: Advisory Board, Research Funding; Gilead: Other: Advisory Board; Bayer: Other: Advisory Board; Zylem LLC: Other: co-founder; research in nanoparticles in cancer.

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Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin

Blood ◽

10.1182/blood.v97.5.1392 ◽

2001 ◽

Vol 97 (5) ◽

pp. 1392-1398 ◽

Cited By ~ 132

Author(s):

Maria-Ana Ghetie ◽

Helen Bright ◽

Ellen S. Vitetta

Keyword(s):

Cell Growth ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

B Lymphoma ◽

B Lymphoma Cells ◽

Anti Cd20

In 1997, a chimeric anti-CD20 monoclonal antibody (mAb) (Rituxan) was approved for the treatment of low-grade/follicular B-cell lymphoma. Rituxan has a long half-life and low immunogenicity, and it mediates effector function. Rituxan induces apoptosis in some tumor cell lines in vitro. Previous studies with mAbs that react with neoplastic B cells have demonstrated that homodimers of immunoglobulin G ([IgG]2) often inhibit cell growth more effectively than their monomeric (IgG)1counterparts. In this study, the ability of IgG or F(ab′)2 homodimers vs monomers of Rituxan were compared for their ability to inhibit the growth of several different B-lymphoma cell lines in vitro. It was found that homodimers of Rituxan had superior antigrowth activity in vitro and that F(ab′)2 homodimers were the most active. Homodimers, but not monomers, of Rituxan induced both apoptosis and necrosis of several B-cell lymphoma lines in vitro; the inhibition of cell growth was not dependent upon the presence of Fc receptors or upon 10-fold or greater differences in the density of CD20 on the target cells. Rituxan homodimers, compared with monomers, also rendered drug-resistant CD20+ B-lymphoma cells more sensitive to chemotherapeutic agents and synergized with an anti-CD22 immunotoxin in vitro.

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B-Cell Lymphoma Cells Overexpressing BMI-1 Are Correlated with Drug Resistance through Enhanced Expression of Survivin and Are Effectively Eliminated by T Cells with Anti-CD38 Chimeric Receptor

Blood ◽

10.1182/blood.v116.21.3613.3613 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3613-3613

Author(s):

Joyeeta Bhattacharyya ◽

Keichiro Mihara ◽

Motoaki Ohtsubo ◽

Shin'ichiro Yasunaga ◽

Yoshihiro Takihara ◽

...

Keyword(s):

Drug Resistance ◽

T Cells ◽

B Cell ◽

Cell Lymphoma ◽

Survivin Expression ◽

B Cell Lymphoma ◽

Drug Resistant ◽

Lymphoma Cells ◽

Anti Cancer

Abstract Abstract 3613 BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas. Disclosures: No relevant conflicts of interest to declare.

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Panobinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) In Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines

Blood ◽

10.1182/blood.v116.21.3936.3936 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3936-3936 ◽

Cited By ~ 3

Author(s):

Francisco J. Hernandez-Ilizaliturri ◽

Cory Mavis ◽

Ilir Maraj ◽

Mohammad Muhsin Chisti ◽

John Gibbs ◽

...

Keyword(s):

Western Blot ◽

B Cell ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Anti Tumor Activity

Abstract Abstract 3936 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Panobinostat (LBH589) is a novel and potent DAC class I and II inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant B-cell lymphoma., We studied the anti-tumor activity of panobinostat as a single agent or in combination with the proteasome inhibitor (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed B-cell lymphoma. In addition, we characterized the mechanisms responsible for panobinostat anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of panobinostat (0.5-5nM) +/− BTZ (1-5nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Subsequently, protein lysates were isolated from panobinostat +/− BTZ exposed cells and changes in members of Bcl-2 family proteins were evaluated by Western blot. Finally, to characterize panobinostat's mechanisms-of-action, lymphoma cells were exposed to panobinostat with or without pan-caspase (Q-VD-OPh, 5mM) or autophagy (3-methyladenine [3MA] 5mM) inhibitors and changes in cell viability were detected as above. Optimal experimental conditions were confirmed by Western blot. Panobinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=25). In addition, synergistic activity was observed by combining panobinostat with BTZ in vitro. The pharmacological interactions between panobinostat and proteasome inhibitor could be explained in part by the effects each agent has on the expression levels of Bcl-2 family members. In vitro exposure of lymphoma cells to panobinostat resulted in Bcl-XL down-regulation, whereas BTZ exposure causes up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished panobinostat anti-tumor activity in RSCL but not in RRCL. On the other hand, exposure of RRCL to 3MA, significantly inhibited the anti-tumor activity of panobinostat in RRCL. Together this data suggest that, panobinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and -independent pathways. Our data suggests that panobinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of a proteasome inhibitor (BTZ). A better understanding in the molecular events (caspase-dependent and -independent) triggered by panobinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these exciting agents in future clinical trials. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute) Disclosures: No relevant conflicts of interest to declare.

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Comprehensive in Vitro and in Vivo Analysis of Phosphatidylinositol-3-Kinase Delta (PI3Kδ) Inhibition Using GS-1101 (CAL-101) in Combination with Mammalian Target of Rapamycin (mTOR) Inhibition Using Temsirolimus or Everolimus in Mantle Cell Lymphoma (MCL)

Blood ◽

10.1182/blood.v120.21.3719.3719 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3719-3719

Author(s):

Paul M. Barr ◽

Francisco J. Hernandez-Ilizaliturri ◽

Thomas Murante ◽

Shannon P. Hilchey ◽

Derick R Peterson ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Mtor Inhibitors ◽

B Cell Lymphoma ◽

Mtor Inhibition ◽

Sequential Administration

Abstract Abstract 3719 The clinical efficacy of mTOR inhibition in MCL is limited by known resistance pathways mediated through IRS-1 and mTORC2. Simultaneous inhibition of other molecules downstream of the B cell receptor, such as PI3Kδ, may abrogate such negative feedback mechanisms. PI3Kδ inhibition using GS-1101 has demonstrated early efficacy in MCL. Taken together, the combination of mTORC1 and PI3Kδ inhibition may represent a rationale combination to test in MCL. To this end, we utilized a panel of B cell lymphoma lines including established MCL cell lines (Granta, Jeko, Mino, Rec-1, HBL-2, Z-138), cytarabine resistant MCL lines (MinoAraCR, JekoAraCR, Rec-1AraCR, HBL-2AraCR) and primary MCL cells isolated from patients. In all cell lines, dose-finding experiments using GS-1101 and the mTOR inhibitors temsirolimus and everolimus were performed in triplicates. Cell viability was determined using an Alamar Blue reduction assay. Proteins downstream of PI3K – mTOR signaling were evaluated by western blot analysis. Synergy between the agents was evaluated using Laska et al's model–free test. For in vivo studies, severe combined immunodeficiency mice were injected with 10×106 Z-138 cells on day 0. GS-9820, a PI3Kδ inhibitor optimized for murine studies, was used in lieu of GS-1101. Upon detection of tumor engraftment, animals were divided into 6 groups, each containing 5 mice; Control, GS-9820 at 10 and 20mg/kg/dose, temsirolimus at 10 and 20mg/kg/dose, and GS-9820 plus temsirolimus at 10mg/kg/dose each. GS-9820 was administered by gastric lavage twice daily on days +15 to +19 and +22 to +26. Temsirolimus was administered via tail vein injection on days +15, +17, +19, +22, +24, and +26. Tumor measurements were used to determine therapeutic activity. The initial screen of lymphoma histologic subtypes demonstrated that cell viability was reduced across Burkitt, diffuse large B cell and MCL lines exposed to GS-1101. In MCL lines, the cell viabilities observed after 48 h treatments with GS-1101 (5uM) were 80% ± 6.9, 66% ± 2.2 and 68% ± 4.7 in Granta, Jeko and Rec-1 cells respectively. No difference was observed in cytarabine resistant cells suggesting non-cross resistance with cytarabine. The activity in primary MCL cells was similar using GS-1101 (5uM) [viability range 55%-65%] while peripheral blood mononuclear cells (PBMCs) appeared less sensitive to GS-1101 [78% ± 2.4]. Both mTOR inhibitors provided moderate reductions in viability after 48 h exposures. Compared to untreated controls, the viabilities of Granta, Jeko and Rec-1 cell lines after 48 h exposures to temsirolimus (5nM) were 73% ± 1.3, 53% % ± 6.9 and 54% ± 2.0 respectively as well as 68% ± 2.9, 50% ± 7.4 and 55% ± 2.0 respectively after everolimus (5nM). Similar results were observed in primary MCL cells using temsirolimus (5nM) [range 80%-85%] while PBMCs were largely unaffected [90% ± 2.2]. The combination of GS-1101 and either mTOR inhibitor produced largely additive reductions in cell viability. Synergistic interactions were observed in Rec-1 cells for 8 dose combinations of GS-1101 (0.1–5.0uM) and either temsirolimus (1–5nM) or everolimus (1–5nM) (unadjusted p < 0.05 for all 8 combinations). Evidence of synergy was insufficient at any combination after adjustment for multiple comparisons over the 3 cell lines. Sequential administration using 24 h pretreatment with each agent was evaluated; no benefit over simultaneous administration was demonstrated. Consistent with known mechanisms of action, immunoblotting revealed decreased 4EBP1 and S6K phosphorylation with mTOR inhibition while PI3K inhibition consistently decreased Akt phosphorylation. In vivo, GS-9820 appears active in the Z-138 xenografts at early time points. Tumor size was reduced to 60% ± 5.5 of control at day 18 and 23 using either 10 or 20 mg/kg of GS-9820. Testing of GS-9820 in combination with temsirolimus in this model is ongoing. Our findings indicate that PI3Kδ inhibition using GS-1101 and GS-9820 is active in vitro and also in a MCL murine xenograft. GS-1101 in combination with mTORC1 inhibition largely produced additive in vitro anti-lymphoma effects in MCL. Ongoing work is aimed at understanding the differences in molecular events downstream of PI3K and mTOR inhibition comparing Rec-1 cells, where synergy was demonstrated, with other cell lines to provide insight into optimal therapeutic combinations and to determine in which molecularly defined subsets of MCL they may be most active. Disclosures: Johnson: Gilead Sciences: Employment. Lannutti:Gilead Sciences Inc: Employment.

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Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽

10.1182/blood.v122.21.377.377 ◽

2013 ◽

Vol 122 (21) ◽

pp. 377-377 ◽

Cited By ~ 3

Author(s):

Shruti Bhatt ◽

Daxing Zhu ◽

Xiaoyu Jiang ◽

Seung-uon Shin ◽

John M Timmerman ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cd20 Antibody ◽

Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

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Metformin Induces p53-Dependent Mitochondrial Stress in Therapy-Sensitive and -Resistant Lymphoma Pre-Clinical Model and Primary Patients Sample with B-Cell Non-Hodgkin Lymphoma (NHL)

Blood ◽

10.1182/blood.v126.23.4008.4008 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4008-4008

Author(s):

Juan J Gu ◽

Qunling Zhang ◽

Cory Mavis ◽

Myron S. Czuczman ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Board ◽

In Vitro Exposure ◽

Induction Of Apoptosis

Abstract Introduction: Metformin, a guanidine originally derived for Galega officinalis (French lilac), has been widely prescribed to type II diabetics since 1950 and well known for its safety toxicity profile. Recently, metformin use was associated with a decrease in risk cancer development and lower cancer-related mortality among breast, colorectal, prostrate, lung, hepatic and ovarian cancer patients. Our group previously reported that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). The mechanism(s) of action underlying the antitumor effect of metformin remains not to be fully elucidated. Previous research done in our laboratory revealed that metformin inhibited cell proliferation through repressing PCNA and p21 proteins. Here, we report that metformin activates tumor suppressor p53 by suppressing MDMX in pre-clinical lymphoma models. Methods: A panel of rituximab-sensitive (RSCL), rituximab-resistant (RRCL) cell lines and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of metformin (0-64mM). Changes in cell viability were determined by Presto Blue (Sigma) assay in cell lines and Titer Glo in patient samples. Changes in MDMX, MDM2 and p53 expression levels were determined by western blotting after exposure cells to metformin. MDMX-MDM2-p53 interactions and p53 ubiquitination following in vitro exposure to metformin were determined by immunoprecipitation of p53 and probing for MDMX, MDM2, ubiquitin and p53 in RSCL and RRCLs. Loss of Dψm or induction of apoptosis following metformin exposure were assessed by DiOC6 or Annexin V/PI staining and flow cytometry. Oxidative stress induced by metformin was measured by flow cytometry using dihydrorhodamine-123 (DHR-123) dye. Result: Metformin induced a dose-and time- dependent cell death in cell lines and primary patient tumor cells. In vitro exposure of lymphoma cell to metformin resulted in a decrease in MDMX levels. Immunoprecipitation studies demonstrated that following exposure to metformin, MDMX bound less to p53 leading to less p53 ubiquitination. In vitro exposure of RSCL or RRCL to metformin resulted in the expression of p53 regulated BH3 single domain proteins (Noxa and Puma). Moreover, metformin repressed mitochondrial potential, induced reactive oxidative species (ROS) generation and triggered apoptosis. Conclusion: Our data suggests that metformin had anti-tumor activity against RSCL, RRCL and primary tumor cells isolated from lymphoma patients. The down-regulation of MDMX and re-activation p53 function following metformin exposure may contribute to the disruption in the mitochondria potential, generation of ROS and induction of apoptosis observed in our models RSCL and RRCL. Our finding highlights a potential role for metformin in the treatment of B-cell 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 Funds) Disclosures Czuczman: MorphoSys: Consultancy; Boehringer-Ingelheim: Other: Advisory Board; Celgene: Employment; Immunogen: Other: Advisory board.

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