Rituximab-resistant Cell Lines Research Articles

Metformin Affects the Proliferation Cell Nuclear Antigen (PCNA) and p21 Protein Interaction Resulting in Direct Anti-Tumor Activity and Enhances the Cytotoxic/Biological Effects of Chemotherapy Agents or Rituximab in Lymphoma in Vitro and in Vivo Pre-Clinical Models

Abstract Most cancer cells predominantly produce adenosine triphosphate (ATP) by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria. This shift in cellular metabolism is know as the Warburg effect and is primarily observed in rapidly growing tumors including aggressive B-cell lymphoma and is thought to be a consequence of the progression to cancer rather than the cause of it. Altering the glucose metabolism in cancer cells appears to be an attractive strategy in cancer medicine. Previously, we demonstrated that the acquirement of rituximab resistance was associated with an increase in the Warburg effect leading to concomitant chemotherapy resistance in lymphoma pre-clinical models. The use of metformin, an oral biguanide widely used to treat insulin resistance conditions, during chemotherapy has been associated with improved clinical outcomes in solid tumor patients receiving chemotherapy. In a retrospective analysis, we demonstrated 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). In an attempt to characterize the mechanism by which metformin affects the biology of DLBCL, we studied the metabolic and signaling changes in rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) exposed to metformin. A panel of RSCL and RRCL were exposed to metformin. Changes in Ki67, proliferation cell nuclear antigen (PCNA), and its regulator (p21) were determined by flow cytometry or Western blotting respectively. Ki67/PCNA/p21 changes were correlated to cell cycle distribution, cell viability and chemotherapy sensitivity. For in vivo studies, SCID mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on Day 3,7,10,14), metformin (at 2ug/ml in drinking water from day 3 till the end of experiment) or metformin with rituximab. Differences in survival (measured as the time for limb paralysis development) were evaluated by log-rank test between treatment arms. Metformin in combination with rituximab (mean survival not reached at 69+/- 5.3 days) lead to an improved survival than rituximab (mean survival 57.1+/-4.2 days) (P=0.05). In vitro exposure of RSCL/RRCL to metformin inhibited cell proliferation (measure by, MTT assay, alamar blue reduction, cell titer glow assay, and ki67 staining) in a dose-dependent manner and enhanced the anti-tumor activity of chemotherapy drugs. Perhaps related to this effect, metformin exposure induced G1 phase cell cycle arrest in both RSCL and RRCL. Using the flow cytometry technology (FITC-labeled anti-KI67 antibody/PI-DNA staining), we detected a decrease in Ki67 of RSCL/RRCL distributed in the G1 and S phase following metformin exposure. In vitro exposure of RSCL and RRCL to metformin increased p21 and reduced PCNA levels. Immuno-precipitation of p21 following metformin drug exposure increased in the interaction between p21 and PCNA. Our data suggests that metformin inhibits the proliferation of RSCL or RRCL and enhances the anti-tumor activity of chemotherapy agents or rituximab in lymphoma pre-clinical models (in vitro and in vivo). Perhaps related to the biological effects observed, p21 and PCNA, play a pivotal role on the anti-tumor activity of metformin in lymphoma pre-clinical models. 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 Fund) Disclosures No relevant conflicts of interest to declare.

The SMAC Mimetic Lcl-161 Augments the in Vitro and in Vivo anti-Tumor Activity of Rituximab and Chemotherapy in Rituximab Relapse/Refractory Lymphoma Models

The addition of rituximab to front-line therapy regimens in diffuse large B-cell lymphoma (DLBCL) has greatly improved clinical outcomes, but is also associated with a disease that is more resistant to salvage chemotherapy in the second-line setting, reinforcing the need for new therapies targeted at the overlapping resistance pathways between rituximab and chemotherapy. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines which exhibited significant concurrent chemotherapy resistance. These multi-therapy resistant cell lines (TRCL) exhibit decreased expression of the pro-apoptotic Bcl-2 family proteins Bak and Bak, along with over-expression of several anti-apoptotic proteins, including the inhibitor of apoptosis proteins (IAP) survivin and livin (determined by Western blot). High IAP expression has been associated with inferior clinical outcomes in a range of hematological malignancies, and solid tumors. To determine the impact of IAP over-expression on TRCL rituximab/chemotherapy resistance we utilized a transient siRNA knockdown of both survivn and livin. TRCLs with livin knockdown had a statistically significant improvement in response to several chemotherapy agents including doxorubicin, vincristine, and the proteasome inhibitor carfilzomib (measured at 48 hours with the Cell Titer-Glo viability assay). These results support livin over-expression as a key lymphoma therapy resistance mechanism, and establish IAPs as potential therapeutic targets.Small molecule IAP inhibitors, like LCL-161 (obtained from Novartis), are chemical mimetics of the endogenous IAP antagonist termed the second mitochondrial inhibitor of caspases (SMAC). Western blot analysis indicated that TRCLs treated in vitro with LCL-161 exhibited a dose dependent decrease in the expression of several IAPs, including livin. In addition, LCL-161 increased rates of TRCL apoptosis, and produced synergistic anti-tumor activity when combined with cytarabine, gemcitabine, and carfilzomib in vitro. LCL-161 also enhanced the ex vivo anti-tumor activity of carfilzomib against primary tumor cells isolated from lymphoma patients with both de novo, and relapse/refractory disease. Cell viability and apoptosis induction were determined at 48 hours with CellTiter-Glo viability assays and flow cytometry respectively. To evaluate the anti-tumor effect of LCL-161 in vivo severe combined immunodeficiency (SCID) mice were inoculated with the TRCL Raji-4RH via tail vein injection (iv), and assigned to observation or treatment arms 7 days after inoculation. Treatments were LCL-161 alone (60mg/kg), the combination of rituximab: 10mg/kg, gemcitabine: 120mg/kg, and vinorelbine: 8mg/kg (RGV), or LCL-161 and RGV together. LCL-161 was administered on day 7 as one dose given p.o. by gavage; RGV was also administered on day 7 as a single i.v. dose given by tail vein injection. Differences in survival (measured as the time to the development of limb paralysis) were evaluated with the Log-rank, Breslow, and Tarone-Ware tests across treatment arms. As a single agent LCL-161 was ineffective in controlling Raji-4RH tumor growth in vivo. However, the combination of LCL-161 with RGV (median survival 133 days) resulted in a statistically significant (P=0.002 with each test) improvement in overall survival when compared to RGV alone (median survival 53 days).In summary, IAPs, especially livin, contribute to rituximab/chemotherapy resistance in relapse/refractory B-cell lymphoma models. However, the IAP inhibitor LCL-161 can disrupt this resistance and augment the effect of chemotherapy in resistant lymphoma cell line models, as well as relapse/refractory lymphoma patient samples. In addition, LCL-161 can improve the anti-tumor activity of the RGV chemotherapy regimen, and increase overall survival in a mouse in vivo model of human rituximab/chemotherapy resistant lymphoma. Our data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of aggressive rituximab relapse/refractory B-cell lymphomas. (Supported by a NHI SPORE Lymphoma grant: 5 P50 CA130805-04, a NIH grant R01 CA136907-01A1 and The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.

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

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.

Vorinostat, a Histone Deacetylase (HDAC) Inhibitor, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Pre-clinical models of chemotherapy resistance and clinical observations derived from the CORAL study suggest that primary refractory/relapsed B-Cell Non-Hodgkin Lymphoma (NHL) is a more aggressive and resistant lymphoma to current available treatments. Pre-clinically, we demonstrated that chemotherapy resistance is associated with a deregulation on the apoptotic machinery rendering lymphoma cells resistant to apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Post-transcriptional histone modification plays an important role in regulating gene transcription, and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including: cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-tumor activity in cancer cells. To this end we studied the anti-tumor activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, Vorinostat) in drug-resistant pre-clinical models.A panel of rituximab and chemotherapy resistant cell lines and primary tumor cells isolated from B-cell NHL patients (N=42: Denovo=27, Relapse/Rafractory=15) were exposed to escalating doses of vorinostat. Changes in cell viability were determined by Cell Titer Glo ® (Promega, Fitchburg, WI) luminescent assays. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from vorinostat exposed cells and changes in members of apoptosis, cell cycle family proteins and the acetylation status of histone H3 were evaluated by Western blotting. In addition, cell lines were pre-exposed to vorinostat for 48 hrs and subsequently plated in 384 well plates and exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine) changes in cell viability were determined by Cell Titer Glo and synergistic activity was evaluated using the Calcusm® software.Vorinostat induced dose-and time- dependent cell death in cell lines and in primary tumor cells. In addition, in vitro exposure of lymphoma cells to vorinostat resulted in an increase in p21, decrease cyclin dependent kinase2 (CDK2) and cyclin E; acetylation of histone H3 and was associated with a G1 cell cycle arrest. As expected, PARP cleavage was not observed in cell lines exposed to vorisnostat at IC50 doses, and Caspase inhibitor experiment further showed that blockage of caspase pathway cannot rescue cells and primary patient tumor samples from vorinostat induced cell death, suggesting that alternative cell death pathways were executed (i.e. irreversible cell cycle arrest). Moreover vorinostat was found to enhances the anti-tumor activity of chemotherapy agents.Our data suggests that vorinostat is active in drug-resistant pre-clinical models and that in cell lines with known defective apoptotic machinery, it can active alternative pathway of cell death. Given the multiple pathways affected by HDAC inhibition, vorisnostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B-cell lymphoma. (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) DisclosuresNo relevant conflicts of interest to declare.

Altered Glucose Metabolism In Rituximab-Chemotherapy Resistant Cells Is a Potential Target For Future Therapeutic Development

Although treatment of lymphoma has greatly improved over recent years, acquired rituximab-chemotherapy resistance is an emerging clinical challenge for physicians treating relapsed/refractory diffuse large B-cell lymphoma and other aggressive histological subtypes. The discovery and characterization of cellular pathways up-regulated or down-regulated in rituximab-chemotherapy resistant B-cell lymphoma cells can potentially identify prognostic biomarkers and/or the development of novel therapeutic strategies. Differences in cellular metabolism exist between normal and malignant cells. Cancer cells are known to have an increase in aerobic glycolysis as a source of adenosine triphosphate (ATP) generation (a.k.a. Warburg effect). Inhibition of glucose uptake or its metabolism has been shown to induce cytotoxicity in solid tumor cancer cells. Therefore, we evaluated differences in the glucose metabolism between rituximab-sensitive (RSCL) or rituximab-resistant cell lines (RRCL) representing aggressive subtypes of B-cell lymphoma. Differences in the baseline cell proliferation rate, ATP generation, lactic acid dehydrogenase (LDH) activity, glucose uptake and lactic acid production between RSCL and RRCL were determined. We then blocked glucose uptake in RSCL and RRCL by exposing these cells to 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor, and measured changes in cell viability, cell cycle distribution, mitochondrial potential and induction of apoptosis using the MTT assay and flow cytometry (propidium iodine [PI], DiOC6, and PI/AnnexinV staining, respectively. Finally, we evaluated the cytotoxic effects of 2-DG in combination with multiple chemotherapy agents in RSCL and RRCL. Significant differences in metabolic rate and glucose metabolism were identified. RRCL had a lower proliferation rate, but alternatively had a higher: 1) intracellular concentrations of ATP, 2) LDH activity, 3) production of Lactate, and 4) glucose uptake/consumption. Together these data suggest that RRCL are more dependent on glucose metabolism for generation of energy (i.e. ATP). In vitro exposure of RRCL, and to a lesser degree of RSCL to 2-DG, resulted in a dose- and time-dependent cell death. As expected, in vitro exposure of RRCL to 2-DG resulted in a decrease in: 1) glucose uptake, 2) lactate production, 3) ATP production and subsequent depletion of ATP intracellular reserves. In addition, 2-DG induced greater G1-phase arrest in RRCL. The baseline mitochondrial potential was higher in RRCL than RSCL and correlated with resistance to multiple chemotherapy agents commonly used in the treatment of B-cell lymphoma. Caspase inhibition did not rescue RRCL to the cytotoxic effects of 2-DG, suggesting the execution of as caspase-independent cell death pathway (i.e. autophagy or senescence). In addition, 2-DG exhibited additive/synergistic effects when combined with doxorubicin in RRCL. In summary, our data suggests that the acquirement of rituximab-chemotherapy resistance in RRCL is associated with deregulation in the mitochondrial potential leading to chemotherapy resistance and an altered cellular metabolism that is primarily dependent on glucose uptake and glycolysis to generate ATP. Developing therapeutic strategies that selectively inhibit glucose uptake or glucose metabolism in cancer cells could prove to be a promising strategy for the treatment of relapsed/refractory lymphoid malignancies in the future. (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: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Alterations In Mitochondrial Priming May Be a Mechanism For Acquired Resistance To Therapy In Diffuse Large B-Cell Lymphoma

Molecular profiling and real-time detection of therapy-resistant lymphoma cells have the potential to improve clinical outcomes by identifying biomarkers capable of predicting treatment efficacy, providing insight into key regulatory signaling pathways suitable for therapeutic targeting, and tailoring treatment selection. Czuczman et al developed and characterized several cell-line models to study the mechanisms involved in the acquirement of rituximab-resistance in B-cell lymphoma. Rituximab-resistant cell lines (RRCLs) were found to be also resistant to multiple chemotherapy agents when compared to rituximab-sensitive cell lines (RSCL). RRCLs have decreased expression of Bax and Bak, key pro-apoptotic members of the Bcl-2 family of proteins. Expression of Bcl-2 family members favoring cell survival has been seen in other lymphoma cell lines that are known to be chemotherapy resistant.It is important to validate the role of Bax/Bak expression in rituximab/chemotherapy resistance in a more clinically relevant setting and to develop assays that detect rituximab/chemotherapy resistant cells in real-time, ideally before therapy is started in a given patient. To this end, we studied 1) correlations in clinical outcomes in lymphoma patients based on pre-treatment Bax/Bak levels an 2) differences in BH3 profiling, a technique that allows for functional assessment of how “primed” cells are (that is, how capable they are of undergoing apoptosis) between RSCL and RRCL.To study the pattern of expression of Bax/Bak in lymphoma and its clinical significance, we retrospectively evaluated differences in the expression these pro-apoptotic proteins in patients evaluated at the Dana-Farber/Brigham and Women's Cancer Center using a tissue micro-array (TMA) containing samples from 180 patients with different subtypes of B-cell lymphoma. Demographic, clinical and pharmacological characteristics were obtained for each patient. Bax/Bak staining was reported as negative, weak, moderate, or intense.In addition, RSCL and RRCL underwent BH3 priming studies. Briefly, RSCL or RRCL were exposed to a panel of BH3 only, pro-apoptotic proteins, and the amount of mitochondrial outer membrane permeabilization (MOMP) was measured over time as previously described.In highly primed cells, MOMP occurs quickly after addition of BH3 peptides, indicating that balance of pro- and anti-apoptotic members of the Bcl-2 family is in favor of apoptosis. This technique permits the assessment of the apoptotic potential in real-time.Generally, there were high levels of Bax and Bak expression amongst the lymphoma specimens represented on the TMA. Of those that could be analyzed, 90% of samples stained moderately or intensely for Bax, and 70% stained moderately or intensely for Bak. Those specimens with decreased expression did not reliably correspond with a more refractory clinical course based on the data available. Ten (10) of the patients included in the TMA had been previously treated with rituximab. Of these patients, 90% stained moderately or intensely for Bax. Nine (9) of these patient samples could be stained for Bak, and 88.9% of them stained moderately or intensely. Significant differences were observed in the BH3 profiling of RSCL and RRCL. Raji 2R and Raji4RH (RRCLs) were less primed than Raji cells (RSCL), suggesting a decrease in the mitochondrial potential.In summary, while Bcl-2 family members play an important role in the development, maintenance, and progression of B-cell lymphoma, the level expression of each individual Bcl-2 family member (i.e. Bax or Bak) appears to have less of an effect on clinical outcome that the overall balance between pro- and anti-apoptotic members. This underscores the need to develop and validate a real-time assay to define the apoptotic threshold of a cancer cell. BH3 priming further demonstrated that RRCL have impaired MOMP. Potentially, this could be adapted for the clinical setting to identify patients most likely to require alternative therapeutic strategies.1Czuczman MS et al. Clin Cancer Res. 2008;14:1561-70.2Olejniczak S. et al. Clin Cancer Res. 2008;14:1550-60.3Deng J et al. Cancer Cell. 2007;12:171-1854 Ryan J, et al. PNAS. 2010; 107(29):12895-12900. Disclosures:No relevant conflicts of interest to declare.

The Acquirement Of Rituximab Resistance Is Associated With De-Regulation Of The PI3K/Akt/mTOR Signaling Pathway Leading To Chemotherapy Resistance In Burkitt Lymphoma Pre-Clinical Models

Treatment with multi-agent chemotherapy regimens has significantly improved survival in pediatric Burkitt Lymphoma (BL) leading to long-term survival in over 80% of cases. The incorporation of rituximab in the treatment of pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been slower than in adults, but may improve clinical outcomes in high risk pediatric BL patients when combined with chemotherapy regimens. On the other hand, BL patients with primary refractory or relapsed disease have a dismal prognosis, stressing the need to identify the mechanism(s) resulting in chemotherapy/rituximab resistance and to develop novel therapeutic approaches. To this end, we exposed a BL cell line (Raji) to escalating doses of rituximab with or without human serum and generated/characterized several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or rituximab-chemotherapy resistant (Raji 2R and Raji 4RH) (RCRCL) cell lines. Subsequently, we screened for aberrant activation of signal transduction pathways between RSCL (Raji), RRCL (Raji 7R and Raji 8RH), or RCRCL (Raji 2R and Raji 4RH) in an attempt to define what pathways were associated with resistance to both rituximab and chemotherapy agents. This was accomplished by the analysis of phosphorylation patterns on key-regulatory members of pre-defined signal transduction pathways using Western blotting, phospho-flow cytometry studies and phosphoproteomics. Effects on cell viability or cell cycle distribution of RRCL or RCRCL following pharmacological inhibition of key-regulatory pathways identified was then performed using the alamar blue reduction assay or flow cytometry respectively. While total Akt (Protein kinase B) expression was similar between all the types of BL cell lines tested, we found that RCRCL (Raji 2R and Raji 4RH) had an increase in basal phosphorylation levels of Akt at the Ser473 and Thr308 phosphorylation sites when compared to RSCL (Raji cells) or RRCL (Raji 7R and Raji 8RH). These findings were confirmed by phospho-flow cytometry studies. Phosphoproteomic analysis comparing Raji (RSCL) against Raji 4RH (RCRCL) cells identified an increase of at least 2 fold in the phosphorylation of 315 proteins in RCRCL including several direct targets of AKT such as GSK3B, WEE1, FOXO1 and PRAS40. Altered phosphorylation of multiple Akt/mTOR downstream proteins (BAD, 4EBP1, GSK3B and ERK) was detected by western blot in RCRCLs (Raji 2R and Raji 4RH) compared to RSCL and RRCLs. In vitro exposure of RCRCL (Raji 2R and Raji 4RH) to escalating doses of MK-2206, a selective Akt inhibitor, or idelalisib, a selective PI3 kinase delta inhibitor, resulted in a dose- and time-dependent decrease in cell viability of RCRCL and to a lesser degree RRCL and RSCL (RCRCL vs. RRCL/RSCL, p<0.05). The IC50 of MK-2206 was lower in RCRCL (Raji-2R = 2.6µM and Raji-4RH = 3.2µM) than in RSCL (Raji= 4.4µM) or RRCL (Raji 7R = 4.0µM and Raji 8RH = 5.2µM). Similarly, the IC50 for idelalisib was lower in RCRCL (Raji-2R = 61µM and Raji-4RH = 149µM) than in RSCL (Raji= 341µM) or RRCL (Raji 7R = 195µM and Raji 8RH = 318µM). In addition, PI3K/Akt/m-TOR inhibition with either MK-2206 or idelalisib induced cell cycle arrest in G1 phase in RSCL/RRCL, but G2/M cell cycle arrest was observed in RCRCL. In BL cells pre-treated with idelalisib (10µM and 50µM) for 24 hours prior to exposure to doxorubicin (1µM, 10µM or 20µM) for 48 hours, the RCRCL Raji 2R exhibited an increased sensitivity to doxorubicin compared to non-idelalisib exposed controls (untreated vs. idelalisib 10µM vs. idelalisib 50µM: doxorubicin 10µM=78% vs. 69% vs. 56%, p<0.05; doxorubicin 20µM=61% vs. 49% vs. 43%, p<0.05). Raji and Raji 7R cells pre-treated with idelalisib did not exhibit an increase in doxorubicin sensitivity. Together our data suggest that constitutive phosphorylation/activation of the PI3K/Akt signal transduction pathway is associated with the development of resistance and may play a role in shared resistant pathways that lead to the acquirement of chemotherapy resistance observed in some rituximab-resistant cell lines. Additionally, inhibition of the PI3K/Akt/mTOR pathway may partially re-sensitize chemotherapy resistant cells to the cytotoxic effects of chemotherapeutic agents. Targeting the PI3K/Akt/mTOR signaling pathway may be clinically relevant in some patients with relapsed/refractory BL. (Research supported by a grant from Hyundai Hope on Wheels and a St. Baldrick's Foundation Scholar Award) Disclosures:Czuczman:Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

CBL-C137, a Curaxin and Potent Suppressor Of NF-Kb Activity and Modulator Of p53 Function Is Active In Rituximab-Sensitive Or Resistant Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) Cells

The identification of critical signaling pathways necessary for the development, maintenance and progression of specific subtypes of DLBCL are necessary in order to develop novel therapeutics against them. Increased NFkB activity and p53 deregulation contribute to either the pathogenesis of some types of DLBCL (i.e. activated B-cell [ABC] subtype) or to rituximab and/or chemotherapy resistance in B-cell lymphoma. Optimal targeting of NFkB activity is an attractive therapeutic strategy that has been evaluated in pre-clinical and clinical models for over a decade with variable degrees of success. CBL-C137 is a novel and potent member of the curaxin family, capable of modulating p53 and NFkB activity and inducing cell death in several solid tumor cancer models. It has never been previously studied in lymphoid malignancies. In order to study and define the therapeutic potential of curaxins in B-cell lymphoma, we evaluated CBL-C137 in lymphoma pre-clinical models. A panel of rituximab sensitive or resistant lymphoma cell lines representing the two most common subtypes of DLBCL (i.e. ABC-DLBCL and germinal center B-cell [GCB] DLBCL) were exposed to CBL-C137 (0.5-16mM). Changes in cell viability; cell cycle distribution; apoptosis and p53/ NFkB p65 expression were evaluated by measuring ATP content, flow cytometry, and Western blotting, respectively. Subsequently, GCB- or ABC-DLBCL cells were exposed to CBL-C137 alone or in combination with various chemotherapy agents or other available (but less selective) NFkB inhibitors (i.e. lenalidomide or Ibrutinib) for 24 or 48 hrs. Changes in cell viability were determined using the cell titer glo assay. In addition, we conducted standardized 51Cr release assays on cells previously exposed to either CBL-C137 or DMSO to investigate the effects of NFkB inhibition on rituximab (or other anti-CD20) antibody-associated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC). CBL-C137 induced dose- and time- dependent cell death in ABC-DLBCL greater than in GCB-DLBCL cell lines. The IC50 for CBL-C137 in ABC-DLBCL (rituximab/chemotherapy sensitive or resistant cells) ranged from 1.36 to 2.77mM. In contrast rituximab/chemotherapy GCB-DLBCL cells exhibited the highest IC50 (11.91mM, 95% C.I 7.1-19.8mM). In sensitive DLBCL cells, CBL-C137 induced both apoptosis and cell cycle arrest in G1 phase. Moreover, in vitro exposure to CBL-C137 decreased p53 and p65 in sensitive cells. CBL-C137 increased Lenalidomide, but not Ibrutinib, anti-lymphoma activity in the conditions tested. Finally, CBL-C137 did not affect rituximab or other anti-CD20 antibody-associated ADCC or CMC in DLBCL cells. Our data suggest that CBL-C137 is active in DLBCL pre-clinical models, primarily in ABC-DLBCL cell lines. In sensitive cells, CBL-C137 modulates p53 and NFkB activity and promotes death and/or cell cycle arrest. Ongoing studies are aimed to further define the anti-tumor effects of CBL-C137 in combination with other small molecules inhibitors targeting directly or indirectly NFkB activity in lymphoma. (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:Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

The novel proteasome inhibitor carfilzomib induces cell cycle arrest, apoptosis and potentiates the anti‐tumour activity of chemotherapy in rituximab‐resistant lymphoma

Targeting the proteasome system with bortezomib (BTZ) results in anti-tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B-cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre-clinical models. Rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), and primary tumour cells derived from B-cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP-cleavage and/or changes in Bcl-2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose- and time-dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase-dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro-apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre-clinical data supports future clinical evaluation of CFZ in B-cell lymphoma.

Abstract 3378: The rGel/BLyS fusion toxin can overcome chemoresistance in malignant B cells..

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive sub-type accounting for 30-40% of non-Hodgkin's lymphoma. Approximately 95% of B cell lymphoma expresses CD20 and 26% of relapsed B-cell lymphoma patients demonstrate reduced CD20 expression on tumor cells after treatment with the anti-CD-20 antibody rituxumab. This result suggests that novel agents that can overcome rituximab resistance may be an excellent candidate for the treatment of DLBCLs which are resistant to rituximab. MCL and ABC-DLBCL cell lines (SP53, Mino, OCI-Ly3, OCI-Ly10 and U2932) were resistant to rituximab whereas GC-DLBCL cell lines (SUDHL-4 and SUDHL-6) were very sensitive to rituximab. To understand the mechanism of rituximab resistance, we developed rituximab-resistant SUDHL-6 cell line. Compared with parental rituximab-sensitive cells (SUDHL-6-S, IC50=0.2 μg/ml), rituximab-resistant cells (SUDHL-6-R, IC50 &amp;gt; 1,000 μg/ml) demonstrated ∼5,000 fold resistance. The rituximab-sensitive cells formed single-cell suspensions whereas resistant cells demonstrated cell aggregates. We previously generated a highly cytotoxic fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B cells. The rituximab-sensitive (SUDHL-6-S) and resistant (SUDHL-6-R) both cells showed similar IC50 values (150 nM) for rGel/BLyS. In contrast, intrinsically rituximab-resistant cell lines (SP53, Mino, OCI-Ly3, OCI-Ly10 and U2932) were also sensitive to rGel/BLyS. Treatment with rituximab specifically induced down-regulation of p-Akt (Ser473), Akt, and Syk and activation of caspase-9, caspase-3 and PARP in a dose-dependent manner in rituximab-sensitive cells (SUDHL-6-S) but not rituximab-resistant cell lines (SUDHL-6-R, OCI-Ly3, or OCI-Ly10). The interactions of marrow stromal cells (MSCs) with tumor cells appear to be important in the development of tumor drug resistance through complex cytokine and adhesion-mediated mechanisms. Treatment with rGel/BLyS induced down-regulation of p-Akt, Akt, Syk, interleukin-6 receptor, Mcl-1, and c-Myc in rituximab-resistant DLBCL cells in both the absence and presence of MSCs. In addition, treatment of cells with rGel/BLyS induced apoptosis through caspase-9 activation, caspase-3 activation, and PARP cleavage. These results suggest that the Akt pathway is involved in rituximab resistance and rGel/BLyS can overcome stromal cell-mediated drug resistance which is a factor reducing the effectiveness of other conventionally-used agents for the treatment of DLBCLs which are resistant to rituximab. This research was conducted, in part, by the Clayton Foundation for Research. Citation Format: Mi-Ae Lyu, Lawrence H. Cheung, John W. Marks, Michael G. Rosenblum. The rGel/BLyS fusion toxin can overcome chemoresistance in malignant B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3378. doi:10.1158/1538-7445.AM2013-3378

Abstract A19: Significantly targeting rituximab resistant B-cell lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR) modified expanded natural killer (NK) cells.

Abstract Background: The outcome for children and adolescents with BL has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Czuczman et al generated Rituximab resistant BL cell lines, which provided a good model to study mAb resistance and design novel therapies (Czuczman et al, Clinical Cancer Research, 2008). Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Efficient expansion of primary NK cells with K562-mbIL15-41BBL and retargeting expanded NK cells with Anti-CD20 CAR by retrovirus were reported by our group previously (Chu &amp; Cairo, et al, ASH, 2011). Objective: To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-viral method and to apply these modified NK cells on antibody resistant BL, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR+) modified PBNK cells following mRNA nucleofection against rituximab resistant BL cells. Methods: PBMC were expanded with inactivated K562-mbIL15-41BBL cells for 7 or 14 days. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab')2 fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay against CD20+ Ramos, Daudi, Raji, two Rituximab resistant cell lines: Raji-2R and Raji-4RH, and CD20- control cells: RS4;11 and Jurkat. CD107a degranulation in exPBNK was measured by flow cytometry. Results: CD56+CD3- exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15-41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56+CD3-. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection. CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors or inhibitory receptors. exPBNK in vitro cytotoxicity was significantly enhanced by CAR+ exPBNK compared to CAR- exPBNK against CD20+ BL at E:T=10:1 (n&amp;gt;3, p&amp;lt;0.001): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p&amp;lt;0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%), Raji (34.15+ 1.13% vs 6.85+ 2.04%), Raji-2R (43.59+ 1.2% vs 21.34+ 1.75%), and Raji-4RH (37.66+ 1.77% vs 13.25+ 0.7%). However, there was no significant difference against CD20- RS4;11 or Jurkat cells. Consistently, CD107a degranulation was enhanced in CAR+ exPBNK compared to CAR- exPBNK in response to CD20+ Ramos, however, there was no significant difference in response to RS4;11 or medium. Conclusion: Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20+ BL and Rituximab resistant cell lines associated with a significant increase in CD107 degradulation. Future directions include examining CAR+ exPBNK cytotoxic activity against CD20+ resistant tumor cells from patients and testing the anti-tumor activity of CAR+ exPBNK against resistant cells in xenograft mice. Citation Format: Yaya Chu, Janet Ayello, Ashlin Yahr, Jared Katz, Lowrence Lo, Allyson Flowers, Mitchell S. Cairo. Significantly targeting rituximab resistant B-cell lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR) modified expanded natural killer (NK) cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A19.

AMG 319, a Novel Inhibitor of Phosphoinositide-3 Kinase Delta (PI3Kd), Demonstrates Activity in Lymphoma Pre-Clinical Models

AbstractAbstract 3718The PI3k/AKT/mTOR signaling pathway has been found to be deregulated in patients with various subtypes of B-cell lymphoma including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Targeting this particular pathway has been evaluated in multiple pre-clinical models and clinical trials using pharmacological inhibitors with variable degrees of success. More selective and potent PI3K inhibitors, such as AMG 319 are being developed and need proper evaluation in currently relevant pre-clinical models (i.e. rituximab resistant models). To this end, we evaluated the activity of AMG 319 as a single agent or in combination with monoclonal antibodies, targeted agents and chemotherapy drugs in a panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary lymphoma cells isolated from patients with treatment naïve or relapsed/refractory (rel/ref) B- and T-cell lymphoma. Furthermore, we attempted to characterize the cell death pathways executed following in vitro exposure to AMG 319. RSCL, RRCL, T-cell lymphoma, Hodgkin’s lymphoma (HL) and MCL cell lines were exposed to escalating doses of AMG 319 (0.1 to 100μM). Isolated primary lymphoma cells were exposed to lower doses of AMG 319 (1 and 10μM) as a single agent and in combination with bortezomib (BTZ) (5–10nM). Cell viability was determined utilizing presto blue and cell titer glo assays. 51Cr release studies were conducted to assess the effect of AMG 319 in rituximab or ofatumumab immunological activity. Lymphoma cell lines were exposed to AMG 319 (10μM) or DMSO (0.1%) for 48 hours. Subsequently cells were labeled with 51Cr and exposed to rituximab, ofatumumab or isotype control with human serum (25%) or effector cells isolated from healthy donors (effector/target ratio 40/1). Finally, to characterize the contribution of the intrinsic apoptotic pathway to AMG 319 activity, primary tumor cells isolated from lymphoma patients were exposed to AMG 319 with or without a pan-caspase inhibitor (Q-VD-Oph, 5μM) and changes in cell viability were detected. AMG 319 exhibited dose-dependant activity as a single agent against various cell lines including RSCL, RRCL, MCL and T-cell lymphoma cell lines. In addition, AMG 319 induced cell death in primary tumor cells (N=5) at lower doses than in cell lines. Preliminary experiments suggest a synergistic activity when AMG 319 is combined with BTZ in vitro, although further analysis is required with a larger number of primary tumor cell samples. Pre-exposure of one RRCL and one RSCL to AMG 319 enhanced the biological activity of rituximab and ofatumumab in terms of antibody dependent cellular cytotoxicity (ADCC) or complement mediated cytotoxicity (CMC), and further investigations with additional cell lines are ongoing. Finally, caspase inhibition diminished AMG 319 activity in primary tumor cells in vitro, suggesting that induction of apoptosis via the mitochondrial pathway plays a role in its anti-tumor activity. Our data suggests that AMG 319 as a single agent is active against NHL cells and potentiate the anti-tumor activity of BTZ and to a lesser degree monoclonal antibodies targeting CD20 antigen. Ongoing studies are aimed to evaluate AMG 319 in combination with chemotherapy agents in order to better optimize the activity of this novel PI3 kinase delta inhibitor in B-cell and T-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

The SMAC Mimetic Lcl-161 Demonstrates Anti-Tumor Activity As a Single Agent and Decreases the Apoptotic Threshold of Chemotherapy Agents in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models

Abstract 61The loss of response to apoptotic stimulus in lymphoma is a major obstacle in the treatment of primary and refractory B-cell malignancies. While the role of the anti-apoptotic Bcl-2 family proteins in the pathogenesis, maintenance, and progression of many sub-types of B-cell lymphoma is well characterized, the impact of the expression level(s) of other key regulatory proteins of cell death pathways (i.e. inhibitor of apoptosis [IAP] proteins) is less defined. The role of IAP proteins in the acquirement of resistance to rituximab or chemotherapy in B-cell lymphoma is unclear. Overexpression of IAP proteins and loss of expression of its antagonist, the second mitochondria-derived activator of caspases (SMAC) correlates with inferior clinical outcomes in a range of malignancies. Perhaps related to the acquisition of resistance, we found that rituximab-resistant cell lines (RRCL) have a deregulation of pro-apoptotic (Bak/Bax) and anti-apoptotic (Mcl-1, Bcl-XL) Bcl-2 family protein expression along with increased expression of the IAP protein survivin. Small molecule SMAC mimetics like LCL-161 are promising agents for lowering the threshold of tumor cell apoptosis, and represent a potential new avenue of therapy for de novo and refractory drug-resistant lymphoma. To this end, we evaluated the anti-tumor activity of LCL-161 in a range of rituximab-sensitive (RSCL), RRCL, and primary lymphoma cells. Cells were exposed to escalating doses of LCL-161 alone or in combination with various chemotherapy agents (i.e. etoposide, doxorubicin, vincristine, gemcitabine, carboplatin, oxaliplatin, bortezomib and cytarabine) for 48 and 72 hrs. Changes in cell viability and ATP content were determined by the CellTiter-Glo viability assay. Protein lysates were obtained from RSCL and RRCL to determine baseline levels of IAP protein family members. LCL-161 displayed significant anti-tumor activity against Burkitt's lymphoma (BL), diffuse large B-cell (DLBCL) and mantle cell lymphoma (MCL) cell lines. Activity was observed in both RSCL and RRCL cell lines. IC50 values for LCL-161 alone were between 35uM and 45uM for the DLBCL lines. Responses were slightly lower in BL and MCL compared to DLBCL cell lines. Synergistic activity between LCL-161 and several chemotherapy agents (e.g. gemcitabine, cytarabine, carboplatin, vincristine, etoposide and bortezomib) commonly used in the management of aggressive lymphoma was seen at physiologically-relevant doses. In vitro exposure of lymphoma cells to LCL-161 decreased the cytotoxic threshold of chemotherapy by 50%, while ex vivo studies with primary patient lymphoma samples showed a decrease of nearly 60%. In vivo studies using a xenograft SCID murine model are planned. In summary, LCL-161 has shown activity both as a single agent, and when combined with several chemotherapy agents in BL, MCL, and DLBCL cell lines as well as primary patient samples. Additionally, LCL-161 exhibits significant cytotoxic activity against RRCLs suggesting an ability to antagonize IAP proteins. This data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of de novo and refractory aggressive B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Effect of obinutuzumab (GA101), a type II glycoengineered monoclonal antibody targeting CD20, against rituximab resistant and sensitive cell lines in B-cell non-Hodgkin lymphoma.

e21150 Background: : Relapse or insufficient response or resistance to Rituximab (RTX) has evolved as a challenge in therapy of B-cell NHL. RTX in combination with FAB chemotherapy is a safe, well tolerated and associated with &gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). Obinutuzumab (GA101), a novel type II glyco-engineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glycoengineered Fc region has shown to cause significantly enhanced ADCC (Mössner et al, Bld 2010; Niederfellner G. et al, Bld 2011). We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines(RRCL), Raji-2R and Raji-4RH, respectively (generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY. Barth M. et al, ASH 2010). Methods: All cell lines were cultured in RPMI with 10% FBS and incubated with escalating doses of GA101 (1-1000 µg/ml) for 24 hrs (n=10), 48 and 72 hrs (n=6). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. B-Cell Lymphoblastic Lymphoma (BLL) U-698-M cells (CD20+) were used as the positive and Loucy cells (CD20-) (T-ALL) were used as the negative controls. Results: At 48 hrs 100 µg/ml of GA101, Raji and U-698-M demonstrated highest cell death of 33.62±5.7% and 37.8± 3.9%, respectively; while Raji-2R and Raji-4RH cell death was 22.8±1.8% and 10.31±0.81% (p&lt;0.02) and (p&lt;0.001) respectively. At 1000 µg/ml of GA101 at 24 hrs, Raji demonstrated increased cell death 40.27±9.1% compared with Raji2R 14.1±0.07 and Raji4RH 20.3± 3.2 (p&lt;0.001) and (p&lt;0.001) respectively. Conclusions: GA101 represents a promising candidate for treating RTX resistant B-Cell Lymphomas and related CD20+B-Cell malignancies. Further studies should aim to identify novel approaches such as combination with activated NK Cells or chemotherapeutic agents that may enhance or synergize with the efficacy of GA101. Further investigations aim to elucidate the mechanisms responsible for minimal cell death induction on the U698M and Raji cell lines.

Evaluation of MLN4924, an investigational NEDD8-activating enzyme (NAE) inhibitor, as a novel targeted agent with single-agent antitumor activity in mantle cell lymphoma (MCL) and Hodgkin lymphoma preclinical models.

e18539 Background: MLN4924 is a novel, potent, and selective inhibitor of NAE, an essential component of the ubiquitin-proteosome system (UPS). We have previously demonstrated that the UPS plays a pivotal role in the development of rituximab and chemotherapy resistance in B-cell lymphomas. There is a scientific need to target the UPS more efficiently in an attempt to reverse acquired resistance to biological and chemotherapeutic agents in relapsed/refractory aggressive lymphoma. To this end, we studied the anti-tumor activity of MLN4924 in rituximab-chemotherapy sensitive and resistant pre-clinical models. Methods: A panel of Burkitt (BL), diffuse large B-cell (DLBCL), MCL and HL lymphoma cell lines and primary tumor cells isolated from patients with non-Hodgkin lymphoma (NHL) (N=10) were exposed to escalating doses of MLN4924 alone or in combination with a panel of chemotherapeutic agents for up to 72 hrs. Cell viability was determined by alamar Blue reduction or CellTiter-glo assay. Apoptosis was determined by Western blotting. Cell cycle analysis was performed by flow cytometry. Results: MLN4924 demonstrated activity in all cell lines in a time-and dose-dependent manner, including the rituximab/chemotherapy-resistant cell lines. The most potent activity was noted in MCL and HL cell lines (IC50 doses were tenfold lower as compared to DLBCL or BL cell lines). A variable degree of anti-tumor activity was observed in primary tumor cells isolated from NHL patients. Induction of apoptosis was observed in rituximab-resistant cell lines. MLN4924 exhibited synergistic anti-tumor activity when combined with bortezomib, bendamustine, and cytarabine in MCL cell lines. Conclusions: MLN4924 exhibits potent in vitro cytotoxic activity against a variety of human B-cell lymphoma cell lines and primary tumor cells isolated from NHL patients. Significant activity was observed in MCL cell lines. Experiments investigating the in vivo activity of MLN4924 are ongoing. MLN4924 is a highly promising agent for the treatment of relapsed/refractory MCL or HL.

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Abstract Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with &amp;gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p&amp;lt;0.05) and Raji vs Raji-4RH (p&amp;lt;0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 516. doi:1538-7445.AM2012-516

Regulation of CD20 in Rituximab-Resistant Cell Lines and B-cell Non-Hodgkin Lymphoma

The aim of this research was to further investigate the contribution of CD20 antigen expression to rituximab activity and define the mechanisms responsible for CD20 downregulation in rituximab-resistant cell lines (RRCL). Rituximab-sensitive cell lines, RRCL, and primary neoplastic B cells were evaluated by chromium-51 release assays, ImageStream image analysis, immunohistochemical staining, flow cytometric analysis, CD20 knockdown, promoter activity, chromatin immunoprecipitation (ChIP) analysis of CD20 promoter, and CD20 plasmid transfection experiments to identify mechanisms associated with CD20 regulation in RRCL. RRCL exhibited a gradual loss of CD20 surface expression with repeated exposure to rituximab. We identified a CD20 antigen surface threshold level required for effective rituximab-associated complement-mediated cytotoxicity (CMC). However, a direct correlation between CD20 surface expression and rituximab-CMC was observed only in rituximab-sensitive cell lines. CD20 promoter activity was decreased in RRCL. Detailed analysis of various CD20 promoter fragments suggested a lack of positive regulatory factors in RRCL. ChIP analysis showed reduced binding of several key positive regulatory proteins on CD20 promoter in RRCL. Interleukin-4 (IL-4) induced higher CD20 promoter activity and CD20 expression but modestly improved rituximab activity in RRCL and in primary B-cell lymphoma cells. Forced CD20 expression restored cytoplasmic but not surface CD20, suggesting the existence of a defect in CD20 protein transport in RRCL. We identified several mechanisms that alter CD20 expression in RRCL and showed that, whereas CD20 expression is important for rituximab activity, additional factors likely contribute to rituximab sensitivity in B-cell lymphoma.

Ofatumumab demonstrates activity against rituximab‐sensitive and ‐resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B‐cell lymphoma

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane-proximal epitope on CD20. To better define ofatumumab's activity, we conducted pre-clinical studies in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), ofatumumab-exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down-regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab-sensitive or rituximab-resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti-CD20 mAbs.

RILYd4, a Human CD59 Inhibitor, Enhances Complement-Dependent Cytotoxicity of Ofatumumab against Rituximab-Resistant B-cell Lymphoma Cells and Chronic Lymphocytic Leukemia

Ofatumumab is an anti-CD20 antibody recently approved for treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL); it mediates much stronger complement-dependent cytotoxicity (CDC) than rituximab. Human CD59, a key membrane complement regulator that inhibits CDC, is highly expressed in B-cell malignancies and its upregulation is an important determinant of the sensitivity of B-cell malignancies to rituximab treatment. Previously, we have shown that the potent CD59 inhibitor rILYd4 sensitizes rituximab-resistant lymphoma cells to rituximab-mediated CDC. Here, we further investigated whether rILYd4 can sensitize B-cell malignancies to ofatumumab-mediated CDC and whether either ofatumumab-mediated CDC or rILYd4-enhanced ofatumumab-mediated CDC correlates with CD20 or CD59 expression, known biomarkers involved in rituximab activity. Rituximab-resistant cell lines and primary CLL cells were used to investigate the antitumor efficacy of the combination of rILYd4 with ofatumumab or rituximab. Propidium iodide staining or alamarBlue assay were used to evaluate the CDC effect. The levels of CD20 and CD59 on the cell membrane were analyzed by flow cytometry. rILYd4 enhanced CDC effects mediated by ofatumumab or rituximab on rituximab-resistant lymphoma cells and primary CLL cells in vitro. The sensitivity to CDC effects mediated by ofatumumab positively correlated with the ratio of CD20/CD59 and negatively correlated with CD59 levels on CLL cells. The degree to which rILYd4 enhanced CDC correlated positively with the CD59 levels on CLL cells. These data suggest that rILYd4 may enhance the anticancer activity of ofatumumab and rituximab in B-cell malignancies that have relapsed after prior antibody-based therapies.

Ofatumumab, a Fully Human Monoclonal Antibody Targeting CD20, Demonstrates Activity Against and Potentiates the Anti-Tumor Activity of Chemotherapy Agents In Rituximab-Sensitive Cell Lines (RSCL), Rituximab-Resistant Cell Lines (RRCL), Lymphoma Xenografts, and Primary Tumor Cells Derived From Patients with B-Cell Non-Hodgkin Lymphoma (NHL)

AbstractAbstract 3917Treatment with the anti-CD20 monoclonal antibody rituximab (RTX) has become the standard-of-care for the treatment of B-cell non-Hodgkin lymphoma (NHL). Despite revolutionizing NHL therapy, many patients demonstrate resistance de novo or develop resistance to RTX following treatment with RTX-containing regimens and/or RTX-based maintenance schedules. Ofatumumab (OFA) is a new 2nd-generation CD20 mAb targeting a novel membrane-proximal epitope on the CD20 antigen. OFA has been FDA-approved for the treatment of fludarabine- and alemtuzumab-refractory CLL and is being evaluated in several clinical trials in NHL. To better define OFA's activity, we conducted pre-clinical studies comparing OFA vs. RTX in a panel of RSCL, RRCL, primary lymphoma cells (n=10), and in a lymphoma xenograft model. Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed to evaluate differences in activity between RTX and OFA. Lymphoma cells were labeled with 51Cr prior to incubation with RTX or OFA (1 or 10 mg/ml) plus effector cells or human serum respectively. 51Cr-release was measured and the percentage of lysis was calculated. In addition, we evaluated the effect of OFA in the cytotoxic effects of chemotherapy agents (doxorubicin, cisplatin and vincristine) and correlated OFA anti-tumor activity to biomarkers known to affect RTX activity (i.e. CD20, CD55, and CD59 surface expression) using qualitative and quantitative flow cytometry. Competitive binding assays were performed using fluorescent-labeled RTX or OFA. OFA was more potent than RTX in elucidating effective CDC at the doses tested not only in RSCL, but also in all RRCL and in primary tumor cells derived from patients with B-cell lymphoma. OFA and RTX were equally effective in ADCC assays. In RSCL and RRCL, there was a linear decrease in sensitivity to RTX (as evaluated by CDC) with decreasing CD20 expression; in contrast, OFA maintained activity even at the lowest levels of CD20 expression. Furthermore, OFA was active despite high levels of CD59 and CD55. OFA had a higher affinity for CD20 than RTX in RSCL. Pre-incubation of RSCL and RRCL with OFA enhanced the anti-tumor activity of chemotherapy agents as determined by alamar blue reduction. Severe combined immunodeficiency (SCID) mice were inoculated via tail vein with Raji cells (day 0) and assigned to observation versus 4 doses of either OFA or RTX (1 or 10mg/kg/dose). The end point of the study was overall survival. Statistical analysis was performed with Kaplan-Meier survival curves and P values calculated by log-rank test. OFA was more effective in controlling in vivo lymphoma growth than RTX. The median survival for animals treated with OFA (1 or 10mg/kg/dose) [73 days and 78 days] was longer than those treated with RTX [56 days and 61 days] (P=0.04 and P=0.04 respectively). Our data suggest that OFA is more potent than RTX not only in RTX-sensitive but also in RTX-resistant models and potentiates the anti-tumor activity of chemotherapy agents commonly used in the treatment of B-cell NHL. We are continuing our research into defining the mechanisms by which OFA increases the lymphoma cell sensitivity threshold to chemotherapy agents and to novel target-specific small molecule inhibitors. Disclosures:Barth:Genmab: Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding. Mavis:Genmab: Research Funding. Gibbs:Genmab: Research Funding. Deeb:Genmab: Research Funding. Czuczman:Genmab: Research Funding.