Anti-tumor Activity Of Chemotherapy Agents Research Articles

Novel Drug to Target HK2 Overcomes Therapeutic Resistance in Preclinical B-Cell Lymphoma Models

Background: The Warburg's effect is a hallmark in cancer. Hexokinase II (HK2) is a glycolic kinase to phosphorylate glucose to glucose-6-phosphate, which can be utilized as the energy source for cells. HK2 is predominantly overexpressed in cancer cells and has been associated with poor clinical outcomes in cancer patients. Previously, we demonstrated that HK2 mRNA and protein levels were increased in diffuse large B-cell lymphoma patients, and were significantly associated with inferior progression free survival (PFS) and overall survival (OS). In our rituximab and chemotherapy-resistant cell lines (TRCL), we demonstrated an upregulation of HK2 expression level is association with voltage-dependent anion channel (VDAC) resulting in a) repress mitochondrial potential and b) global therapy resistance. Knocking out HK2 overcame the chemo-resistance phenotype observed in TRCL. Here, we designed a novel specific inhibitor of HK2 and tested its activity in lymphoma pre-clinical models. Material and Methods: A HK2 specific inhibitor (HK2-001) was designed and generated according to antisense peptide technology, CABS-docking system and ROSWTTA analyzing. A panel of B-cell lymphoma cell lines including rituximab/chemo-sensitive and resistant cell lines (Raji, Raji 4RH, RL and RL 4RH), diffuse large B-cell lymphoma (DLBCL) (DHL4, DOHH2, TMD8 and U2932) and mantle cell lymphoma (MCL) (HBL2, Granta, Mino and Z138) were used. In addition, primary tumor cells isolated from chronic lymphocytic lymphoma (CLL) or other B-cell lymphoma patients were used. Briefly, cell lines or primary tumor cells were exposed to escalating doses of HK2-001 alone or in combination with different chemotherapy agents (doxorubicin, dexamethasone), targeted agents (i.e. burton tyrosine kinase inhibitors, BH3 mimetics) or monoclonal anti-CD20 antibodies. Cell viability was evaluated using the Presto Blue assay in experiments testing pharmacological interactions between HK2-001 and chemotherapy or targeted agents. All experiments were performed in triplicates in three separate experiments. The coefficient of synergy was calculated using CalcuSyn. Results: In vitro exposure of lymphoma cell lines to HK2-001 induced cell death in a panel of B-cell lymphoma cell lines representing rituximab/chemo-sensitive and resistant lymphoma. In addition, synergistic activity was observed between HK2-001 and doxorubicin, ibrutinib, and Venetoclax. These effects were also observed in primary tumor cells isolated from B-cell lymphoma patients. In addition, HK2-001 potentiated the anti-tumor activity of Rituximab and Obinutuzumab ex vivo. Conclusion: We developed a novel peptide targeting HK2. Pre-clinical studies suggested anti-tumor activity as a single agent. More importantly, HK2-001 enhanced the anti-tumor activity of chemotherapy agents, BTK inhibitors, BH3 mimetics and monoclonal antibodies targeting CD20. HK2-001 is a promising novel therapeutic strategy for against B-cell lymphoma, especially in refractory or relapsed patients. Future studies aimed to evaluate the effects of HKII-001 drug combinations in lymphoma animal models are planned and to evaluate its effects on Chimeric antigen receptor T-cells (CART) or bi-specific antibodies.

BCL-Xl Expression Is Druggable Biomarker Associated with a Poor Clinical Outcome in Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Although first-line chemo-immunotherapy cures a significant number of diffuse large B-cell lymphoma (DLBCL) patients, long term survival is only observed in 30% or 41% of patients treated with second-line therapy followed with high dose chemotherapy and autologous stem cell support (HDC-ASCS) or chimeric antigen receptor anti-CD19 T-cell therapy (CART-19) respectively. The use of third-line therapy with chemotherapy drugs or current available small molecule inhibitors for relapsed/refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) is associated with a poor response rate and a median survival of 6.3 months, stressing the need to identify druggable biomarkers associated with therapy resistance and poor clinical outcomes. Methods: To this end, we identified patients with rel/ref DLBCL that failed at least 2 prior lines of therapy treated at our Institute. Demographic, clinical and pathological characteristics were identified. Archived pathological material from the biopsy performed at the second relapse/progression was obtained for each patient. Using the Nanostring nCounter Analysis System (Nanostring Technologies, Seattle, WA), gene expression profiling (GEP) was performed for each sample as previously described using a custom designed code set containing 770 genes. Analysis and normalization of the raw Nanostring data was performed using nSolver Analysis Software v1.1. Raw counts were normalized to internal levels of 7 reference genes: CNOT2, GAPDH, HPRT1, PHGDH, SUMO2, SYS1 and WDR45L. A background count level was estimated using the average count of the 8 negative control probes in every reaction plus 2 standard deviations. Correlation between clinical outcomes (response rate to third line therapy, progression free-survival [PFS] and overall survival [OS]) and IPI score, clinical characteristics and GEP. Subsequently and based on the GEP results, we evaluated the expression of Bcl-XL and other Bcl-2 family proteins in a panel of DLBCL cell lines. Immuno-precipitation (IP) studies were conducted to determine protein-protein interaction between Bcl-XL and other Bcl-2 related proteins. In addition, cell lines were exposed to A1331852, a novel Bcl-XL inhibitor at different doses alone or in combination with chemotherapy drugs. Cell viability was evaluated using Presto Blue assay and IC50 values were calculated using the GraphPad Prism6 software. Results: A total of 53 rel/ref DLBCL were identified that received third line therapy at our Institute. The median age was 59yrs. The median PFS and OS was 23 months and 21 months respectively. Over-expression of BCL-XL RNA levels was associated with a shorter OS (P=0.015, HR=1.17). Pre-clinically higher levels of Bcl-XL as determined by western blotting were found in lymphoma cell lines resistant to chemotherapy agents in vitro. IP studies demonstrated a strong interaction between Bcl-XL and BIM in chemotherapy resistant cell lines. In vitro exposure of DLBCL cell lines to A1331852 resulted in dose- and time-dependent cell death. Moreover, synergistic activity was observed when A1331852 was combined with chemotherapy drugs (doxorubicin, vincristine and etoposide). Conclusion: Our data suggests that high expression level of BCLxL was associated with inferior prognosis in the refractory and/or relapsed DLBCL patients. Inhibition of Bcl-XL results in cell death of rituximab-chemotherapy resistant lymphoma cell lines and potentiates the anti-tumor activity of chemotherapy agents. Selective targeting of Bcl-XL may be a novel therapeutic strategy for rel/ref aggressive B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Ruxolitinib synergistically enhances the anti-tumor activity of paclitaxel in human ovarian cancer.

Treatment for ovarian cancer remains challenging despite a high initial response rate to first line platinum-taxane treatment. Most patients eventually experience recurrence and require further treatment. Persistent activation of STAT3 is associated with cancer growth and progression and is also involved in cell resistance to platinum and taxane treatment. Targeting JAK/STAT3, therefore, could be a potential novel therapeutic approach for treating advanced and chemoresistant ovarian cancer. We investigated the therapeutic potential of ruxolitinib, a JAK1/JAK2 inhibitor that has been FDA-approved for the treatment of myelofibrosis, to treat ovarian cancer either alone or in combination with conventional chemotherapy agents. We show that ruxolitinib inhibits STAT3 activation and ovarian tumor growth both in ovarian cancer cells and in an ovarian cancer mouse model. In addition, ruxolitinib significantly increases the anti-tumor activity of chemotherapy agents, including paclitaxel, cisplatin, carboplatin, doxorubicin and topotecan in ovarian cancer cells. Evaluation of the combination index (CI) shows that ruxolitinib synergistically interacts with paclitaxel in all three human ovarian cancer cells. Finally, our results demonstrate that combination of ruxolitinib and paclitaxel leads to a greater reduction of tumor growth compared to single treatment of either agent in a tumor mouse model that represents late stage ovarian cancer with peritoneal metastasis and ascites formation. Taken together, our findings provide a foundation for clinical trials with ruxolitinib, either as a single agent or in combination with paclitaxel, for the treatment of recurrent and advanced ovarian cancer.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models.Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (Δψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs.Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole.Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. DisclosuresNo relevant conflicts of interest to declare.

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.

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.

MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B-cell lymphoma preclinical models

To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma.

Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Is Active in B-Cell Lymphoma and Enhances the Anti-Tumor Activity of Rituximab, Chemotherapy Agents and Proteasome Inhibitors

Abstract 1649Deacetylases (DACs) are enzymes that remove the acetyl groups from target protein lysines leading to regulation of gene transcription and other cellular processes. Entinostat is a novel and potent class I DAC inhibitor undergoing pre-clinical and clinical testing. We previously demonstrated that entinostat was active against a panel of rituximab-sensitive (RSCL) and –resistant cell lines (RRCL); and enhanced the anti-tumor activity of chemotherapy agents. In the current work, we studied the mechanisms-of-action of entinostat and its effect on rituximab activity and proteasome inhibition. Our results indicate that entinostat potentiates both rituximab and proteasome inhibitor activity in NHL cells through a dual caspase dependent and independent mechanism-of-action. Studies were conducted in RSCL, RRCL and in primary tumor cells isolated from patients with B-cell non-Hodgkin’s lymphoma (n = 43). For studies with a proteasome inhibitor, cells were exposed to escalating doses of entinostat +/− bortezomib of up to 72 hrs. Studies were repeated with or without a pan-caspase inhibitor (Q-VD-OPh, 5uM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Flow cytometric analysis was used to determine changes in cell cycle. Protein lysates from entinostat +/− BTZ exposed cells were evaluated for changes in members of Bcl-2 and cell cycle family proteins. Microarray analysis was performed to discern differences in gene expression between RRCL and RSCL after incubation with entinostat. Separately, we studied the effects of entinostat in vitro and in vivo on rituximab-associated complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). NHL cells were exposed to entinostat or DMSO 0.01% for 48hrs and then labeled with 51Cr. Labeled target cells were then exposed to rituximab or isotype control (10mg/ml) and human serum (25%) (CMC) or PBMCs (Effector:Target ratio, 40:1) (ADCC). For in vivo studies, 6–8 week old SCID mice were inoculated with 1′106 Raji cells via tail vein injection and after a period of 72 hours animals were divided into six cohorts: control, entinostat alone at 5mg/kg/dose, entinostat alone at 20mg/kg/dose alone, rituximab single agent at 10mg/kg/dose, rituximab and entinostat at 5mg/kg/dose and rituximab + entinostat at 20mg/kg/dose. Entinostat was administered by gastric lavage daily on days +3–8 and +11–16, and rituximab was administered via tail vein injection on days +3, +8, +11, and +16. Difference in survival between treatment groups was performed by Kaplan-Meier analysis. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary lymphoma cells. In vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Synergistic activity was observed by combining entinostat with BTZ but not rituximab in vitro. Microarray data demonstrated that p21 was up-regulated after exposure to entinostat. Additionally, we found an upregulation of ICAM2, down-regulation of E2F2 but no changes in CD20 mRNA. Findings were confirmed at the protein level. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL suggesting that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways (i.e. cell cycle arrest). Furthermore, In vivo treatment of SCID mice with high dose entinostat in combination with rituximab led to prolongation of the mean survival (87 days, 72–103 95% C.I. P=0.05) compared to rituximab (67 days, 52–82 95% C.I.) entinostat 20 mg/kg (19 days, 19–20 95% C.I.) alone. Our data suggests that entinostat is active against RSCL and RRCL and potentiates the anti-tumor activity of BTZ or rituximab. In addition, entinostat appears to have a dual mechanism of action that supports its clinical development in rituximab-sensitive or resistant lymphomas. Ongoing studies are aimed to further define the molecular events responsible for entinostat mechanisms-of-action and optimize its spectrum of anti-tumor activity. (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.

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.

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

AbstractAbstract 3939The 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.

Efficacy of the DAC inhibitor LBH589 in both rituximab-sensitive and rituximab-resistant lymphomas and effect on the antitumor activity of chemotherapy agents, monoclonal antibodies, and bortezomib

8576 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histones (class II)], leading to regulation of gene transcription and other cellular processes. LBH589 is a novel and potent DAC class I and II Inhibitor undergoing pre-clinical and clinical testing. In order to develop therapeutic options for refractory/resistant B-cell lymphomas we studied the effects of LBH589 in the anti-tumor activity of chemotherapy agents and monoclonal antibodies in a panel of rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), and in lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed B-cell lymphomas by negative selection using magnetic beads. NHL cells lines were exposed to the following chemotherapy agents or monoclonal antibodies: CDDP, doxorubicin, vincristine, bortezomib or rituximab, veltuzumab, or isotype, alone or in combination with LBH589. In dose-sequence studies the treatment with LBH589 preceded or followed in vitro exposure to the chemotherapy agent or the monoclonal antibody by 24 hrs. Changes in mitochondrial potential were determined by alamar blue reduction using a kinetic assay. Patient-derived tumor cells were exposed to either LBH589, bortezomib or both. Changes in ATP content were determined by cell titer glow assay. RNA was isolated from NHL cell lines exposed to LBH859 and changes in gene expression of the Bcl-2 family members were determined by qualitative polymerase chain reaction (PCR). LBH589 was active as a single agent against RSCL, RRCL or patient-derived tumor cells. In addition, Bcl-XL gene down-regulation was observed following exposure to LBH859. Synergistic activity was observed by combining LBH589 and chemotherapy agents, bortezomib or either of the two anti-CD20 mAbs studied. The sequence of administration impacted the degree of antitumor activity observed. Our data suggests that LBH589 is active against various RSCL, RRCL and patient-derived tumor cells. Findings suggest that LBH589 added to systemic anti-CD20 and/or chemotherapy could result in a novel and potent treatment strategy against B-cell lymphomas. No significant financial relationships to disclose.

The DAC Inhibitor LBH589 Is Highly Effective in Both “Therapy”-Sensitive and -Resistant Lymphomas and Enhances the Anti-Tumor Activity of Chemotherapy Agents, Monoclonal Antibodies, and Bortezomib

Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Histones are one of the target proteins, which is why DACs (Class I) are sometimes referred to as histone deacetylases (HDACs). DACs also target non-histone proteins, which include transcription factors, a-tubulin, and HSP90 (Class II DACs). LBH589 is a novel and potent DAC Inhibitor that inhibits both class I and II of DAC activity, inducing cell death in tumor cells undergoing pre-clinical and clinical testing. Other investigators had demonstrated that DACs can down-regulate Bcl-2 expression in lymphoma cells (Duan, Heckman, Mol Cell Biol. 2005; 25(5):1608-19), and potentially overcome chemotherapy resistance in B-cell lymphomas. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphomas we studied the effects of LBH589 at various doses (2nM to 20nM) and schedules of administration (before, after or concurrent administration) in the anti-tumor activity of chemotherapy agents and monoclonal antibodies in a panel of rituximab-sensitive (RSCL) (Raji and RL cells) and rituximab-resistant cell lines (Raji 2R, Raji 4RH and RL-4RH) (RRCL). In addition, we utilized lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed B-cell lymphomas. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells lines were exposed to the following chemotherapy agents or monoclonal antibodies: CDDP (1 to 100mM), Doxorubicin (4 to 16mM), Vincristine (1 to 5mM), Bortezomib (1 to 10nM) or Rituximab (10mg/ml), A20 (10mg/ml), or Trastuzumab (Isotype, 10mg) alone or in combination with LBH589. In dose-sequence studies the treatment with LBH589 preceded or followed in vitro exposure to the chemotherapy agent or the monoclonal antibody by 24 hrs. Changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 15 minute intervals for 24 and 48 hrs. Patient-derived tumor cells (10 samples up to date) were exposed to either LBH589, bortezomib or both at different doses. Changes in ATP content were determined using the cell titer glow assay. LBH859 was active as a single agent against rituximab-sensitive or resistant cells tested as well as in tumor cells derived from lymphoma patients. Significant anti-tumor activity was more pronounced after 48 hrs of drug exposure even at 2nM doses of LBH859. In addition, significant synergistic activity was observed by combining LBH589 and chemotherapy agents (CDDP, doxorubicin or Vincristine), bortezomib or the two mAbs targeting CD20 studied (rituximab and veltuzumab). The sequence of administration impacted the responses observed with bortezomib and mAbs, but not the chemotherapy agents evaluated. Pre-incubation of NHL cells with LBH589 prior to bortezomib or mAb therapy leads to the maximum optimization of each agent than another sequence of administration. In summary, our data suggests that LBH589 is active against various RSCL, RRCL and patient-derived tumor cells and potentiate the anti-tumor effects of chemotherapy agents, mAbs targeting CD20, and bortezomib. Findings suggest that LBH589 added to systemic anti-CD20 and/or chemotherapy could result in a novel and potent treatment strategy against B-cell lymphomas.

Targeting BH3-Domain Anti-Apoptotic Proteins with ABT-737 Decreases DNA Synthesis, Induces Cell Death and Sensitizes Rituximab-Sensitive and Rituximab-Resistant Non-Hodgkin's Lymphoma Cell Lines to the Anti-Tumor Activity of Chemotherapy Agents.

ABT-737 is a potent pan-bcl-2 inhibitor with known activity against chronic lymphocytic leukemia (CLL) cells and Mantle cell lymphoma (MCL) cell lines, currently undergoing phase I testing. Bcl-2 over-expression is associated with chemotherapy resistance and correlates with a poor clinical outcome in diffuse large B-cell lymphoma (DLBCL). Recently, we demonstrated that rituximab resistance is associated with deregulation of BH3-domain pro and anti-apoptotic proteins leading to concomitant resistance to several chemotherapy agents. Targeting BH3-domain anti-apoptotic proteins with ABT-737 is an attractive strategy to circumvent/overcome both acquired antibody and chemotherapy resistance. To this end, we studied the effects of ABT-737 in combination with rituximab or chemotherapy agents in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). NHL cell lines were exposed to escalating doses of ABT-737(01, 0.01, 0.1, 1, 10, 20, 50 and 100 mM); changes in mitochondrial potential and ATP production were determined by alamar blue reduction or celltiter-glo® luminescent cell viability assays at different time periods, respectively. Once the optimal dose and time of ABT-737 exposure was determined, cell growth inhibition and immunological assays were conducted to determine the effects of BH3-domain targeting on rituximab activity utilizing standardized immunological assays evaluating changes in DNA synthesis, as well as rituximab-mediated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC). In addition, lymphoma cells were exposed in vitro to ABT-737 (0 to 100mM) with or without CDDP (0 to 10mM) or Doxorubicin (0 to 1mM). Following a 24 and a 48 hour-period of drug exposure, induction of apoptosis was determined by caspase-3 activity assays. ABT-737 induced a dose-dependent cell death in various NHL cell lines. Anti-tumor activity correlated directly with baseline Bcl-2 and inversely to Mcl-1 levels. Up to 75% of cell death was observed in all cell lines exposed to 10–100mM of ABT-737. Pre-incubation of NHL cells to ABT-737 at 10mM enhanced rituximab-mediated CMC. The mean percentage of rituximab-associated CMC on vehicle pre-treated Raji cells was 18% versus 30% for Raji cells exposed to ABT-737. Rituximab-associated ADCC was not affected by ABT-737. In addition, in vitro exposure of NHL cells to ABT-737 in combination with either CDDP or Doxorubicin induced a three fold increase in caspase-3 activity as compared to that achieved by each chemotherapy agent alone. In summary, our data strongly suggests that ABT-737 is active against various RSCL and RRCL and augments the anti-tumor activity of rituximab and chemotherapy agents. Ongoing studies in lymphoma xenografts and fresh primary lymphoma patient samples will further validate the use of ABT-737 in combination with rituximab and/or chemotherapy and give further support for their use in clinical trials.

Targeting BH3-Domain Anti-Apoptotic Proteins with GX15-070 Decreases DNA Synthesis, Induces Cell Death and Sensitizes Rituximab-Sensitive and Resistant Non-Hodgkin's Lymphoma Cell Lines to the Anti-Tumor Activity of Chemotherapy Agents.

GX15-070 is a potent pan-bcl-2 inhibitor with known activity against chronic lymphocytic leukemia (CLL) cells and mantle cell lymphoma (MCL) cell lines, currently undergoing phase I testing. Bcl-2 over-expression is associated with chemotherapy resistance and correlates with a poor clinical outcome in diffuse large B-cell lymphoma. Recently, we demonstrated that acquisition of rituximab resistance is associated with deregulation of BH3-domain pro- and anti-apoptotic proteins. This leads to concomitant resistance to multiple chemotherapy agents. Targeting BH3-domain anti-apoptotic proteins with GX15-070 is an attractive strategy to potentially overcome acquired biologic and/or chemotherapy resistance. To this end we studied the effects of GX15-070 in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. Functional assays (e.g. ADCC and CMC assays) demonstrated a significant decrease in rituximab sensitivity in RRCL. In addition, resistance to chemotherapy agents (CDDP, doxorubicin, vincristine, etc.) was demonstrated in RRCL. Lymphoma cells were exposed in vitro to GX15-070 (0 to 20mM) with or without CDDP (0 to 10mM) or Doxorubicin (0 to 1mM). Following a 24 and a 48 hour period of drug exposure: induction of apoptosis and the percentage of viability was determined by flow cytometric analysis, Western blotting and trypan-blue staining techniques. In addition, changes in DNA synthesis and cell proliferation following drug exposure were performed using standard [3H]-thymidine incorporation assays. GX15-070 induced a dose-dependent cell death and decrease in DNA synthesis in all the cell lines tested (both RSCL and RRCL). Up to 75% of cell death was observed in all cell lines exposed to 20mM of GX15-070. Anti-tumor activity was seen even at the lowest tested dose of GX15-070 (2mM, 20–25% death cells). Incubation of RSCL and RRCL with 2 or 5mM of GX15-070 induced synergistic cytotoxic and anti-proliferative effects when combined with CDDP and doxorubicin. The IC50 of CDPP and doxorubicin was decrease ten-fold by the concomitant in vitro exposure to GX15-070 in all cell lines tested, including RRCL. Our data demonstrates that GX15-070 is active against both RSCL and RRCL and augments the anti-tumor activity of chemotherapy agents. These results strongly suggest that GX15-070 added to systemic chemotherapy is a potentially valuable and novel therapeutic strategy in the treatment of B-cell NHL.