Evaluation of the Anti-Tumor Activity of MLN4924, A Novel NEDD8 Activating Enzyme Inhibitor, in Pre-Clinical Models of Rituximab Chemotherapy-Sensitive or -Resistant B-Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2761-2761
Author(s):  
Natalie M Czuczman ◽  
Matthew J. Barth ◽  
Richa Dwar ◽  
Cory Mavis ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Clinical Models ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Abstract 2761 Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 61-61
Author(s):  
Kyle Runckel ◽  
Cory Mavis ◽  
Joseph Skitzki ◽  
Myron S. Czuczman ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
De Novo ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Abstract 61 The 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.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3734-3734
Author(s):  
Cory Mavis ◽  
Sarah Frys ◽  
Juan Gu ◽  
John Gibbs ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
In Vitro Exposure ◽  
Anti Tumor Activity

Abstract Abstract 3734 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials. Disclosures: Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3939-3939
Author(s):  
Juan Gu ◽  
Patil Ritesh ◽  
Cory Mavis ◽  
George Deeb ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Anti Tumor Activity

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

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3936-3936 ◽  
Author(s):  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Ilir Maraj ◽  
Mohammad Muhsin Chisti ◽  
John Gibbs ◽  
...  
Keyword(s):  
Western Blot ◽  
B Cell ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Anti Tumor Activity

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

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

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

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

The DAC Inhibitor, LBH589, Is Highly Effective in Both Rituximab-Sensitive and Rituximab-Resistant Lymphomas and Enhances the Anti-Tumor Activity of Bortezomib, Other Chemotherapy Agents, and Anti-CD20 Monoclonal Antibodies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2715-2715
Author(s):  
Francisco J Hernandez-Ilizaliturri ◽  
Cory Marvis ◽  
Ilir Maraj ◽  
Mohammad M Chisti ◽  
John Gibbs ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Anti Cd20 ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Abstract 2715 Poster Board II-691 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 better characterize the role of DAC inhibitors in the treatment of refractory/resistant B-cell lymphomas we studied the anti-tumor effect that LBH589 had when used with chemotherapy agents and anti-CD20 monoclonal antibodies against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or refractory/relapsed B-cell lymphoma. Non-Hodgkin's lymphoma (NHL) cell lines were exposed to the following chemotherapy agents or monoclonal antibodies: CDDP, doxorubicin, vincristine, bortezomib versus rituximab or veltuzumab (or isotype control), 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 primary tumor cells (N=25) were exposed to either LBH589 (2-25uM), bortezomib (1 to 10nM) or both. Changes in ATP content were determined by cell titer glow assay. RNA was isolated from NHL cell lines exposed to LBH859 or bortezomib 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 primary tumor cells. In addition, Bcl-XL gene down-regulation was observed following exposure to LBH859. On the other hand, upregulation of Bak and downregulation of Mcl-1 were observed following proteasome inhibition. Synergistic activity was observed by combining LBH589 and chemotherapy agents, bortezomib or either of the two anti-CD20 mAbs studied. In tumor-derived primary cells from lymphoma patients, the combination of LBH589 and bortezomib resulted in significant anti-tumor activity in follicular, Hodgkin and diffuse large B-cell lymphoma. The sequence of administration impacted the degree of antitumor activity observed (ie in general, exposure of tumors cells initially to LBH589, followed by exposure to chemo/mAbs was associated with the greatest degree of anti-tumor activity). Our data suggests that LBH589 is active against various RSCL, RRCL and patient-derived primary tumor cells. Our findings strongly suggest that LBH589 added to anti-CD20 and/or chemotherapy results in a novel and potent treatment strategy against B-cell lymphoma. Research, in part, supported as part of a subproject on NIH PO1 grant CA103985-1 awarded to the Garden State Cancer Center, Belleville, NJ and NHI R-01 grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute Disclosures: No relevant conflicts of interest to declare.

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2732-2732
Author(s):  
Enrico Derenzini ◽  
Ilaria Iacobucci ◽  
Elisa Brighenti ◽  
Federica Cattina ◽  
Richard Eric Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1836-1836
Author(s):  
Christopher N. Ibabao ◽  
Cory Mavis ◽  
Jenny Gu ◽  
Myron S. Czuczman ◽  
Francisco Hernandez
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Clinical Models ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract 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.

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

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

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

scholarly journals 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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5486-5486
Author(s):  
Kai Xue ◽  
Juan Gu ◽  
Cory Mavis ◽  
Francisco Hernandez ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Chemotherapy Resistance ◽  
Non Hodgkin Lymphoma ◽  
Cycle Arrest ◽  
Clinical Models ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract 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) Disclosures No relevant conflicts of interest to declare.

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