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

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.

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

Blood ◽

10.1182/blood.v126.23.2761.2761 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2761-2761

Author(s):

Juan J Gu ◽

Qunling Zhang ◽

Cory Mavis ◽

Myron S. Czuczman ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Board ◽

Synergistic Activity ◽

Targeted Agents ◽

Chemotherapy Agents

Abstract Background: The poor clinical outcomes of patients with aggressive B-cell lymphoma in the post-rituximab era, stress the need to identify and/or optimize novel targeted agents. Several retrospective and prospective clinical studies had demonstrated that C-myc expression correlates with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory diffuse large B-cell lymphoma (DLBCL). To this end, we evaluated the therapeutic effects of targeting C-myc using JQ1, a novel bromodomain inhibitor in rituximab-sensitive or -resistant models. Methods: A panel of rituximab-sensitive (RSCL) or rituximab-resistant (RSCL) cell lines was exposed to JQ1 (0-100 µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently lymphoma cells were exposed to JQ1 or vehicle and various chemotherapy agents such as doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), Ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Results: In vitro exposure of RRCL and to a lesser degree RSCL to JQ1 resulted in a dose- and time-dependent cell death. Strong synergistic activity was observed when JQ1 was combined with doxorubicin, dexamethasone bortezomib or carfilzomib in vitro. Cell cycle analysis demonstrated that in vitro of RSCL or RRCL to JQ1 resulted in G1 cell cycle arrest. Conclusions: In summary, our data suggests that targeting C-myc expression using JQ1 results in anti-tumor activity against RSCL and RRCL. In addition, JQ1 exhibited synergistic activity when combined with chemotherapy agents (doxorubicin or dexamethasone) or targeted agents (bortezomib or carfilzomib). On going studies are aimed to study the mechanisms by which c-myc inhibition results in cell death in RSCL and RRCL. JQ1 is a distinct targeted agent undergoing clinical evaluation in patients with relapsed/refractory lymphomas. Molecular studies dissecting the cellular pathways affected by JQ1 are important in order to further advance the clinical development of c-myc inhibitors in lymphoid malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures Czuczman: Boehringer-Ingelheim: Other: Advisory Board; Immunogen: Other: Advisory board; MorphoSys: Consultancy; Celgene: Employment.

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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 ◽

10.1182/blood.v122.21.1836.1836 ◽

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.

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

Blood ◽

10.1182/blood.v116.21.3939.3939 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3939-3939

Author(s):

Juan Gu ◽

Patil Ritesh ◽

Cory Mavis ◽

George Deeb ◽

John Gibbs ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Anti Tumor Activity

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

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The Novel Proteasome Inhibitor Carfilzomib (CFZ) Potentiates the Anti-Tumor Activity of Chemotherapeutic Agents in Rituximab-Chemotherapy Resistant Lymphoma Through Inducing G2/M Cell Cycle Arrest and Cell Death

Blood ◽

10.1182/blood.v118.21.4970.4970 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4970-4970

Author(s):

Juan Gu ◽

Natalie M Czuczman ◽

Gregory P. Kaufman ◽

Cory Mavis ◽

Francisco J. Hernandez-Ilizaliturri ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Tumor Cells ◽

Primary Tumor ◽

Proteasome Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

M Cell ◽

Anti Tumor Activity

Abstract Abstract 4970 The concept of proteasome inhibition (PI) is an attractive therapeutic approach for resistant/refractory lymphoma. While bortezomib has modest activity against certain types of lymphomas, a significant number of patients develop resistance or experience dose-limiting toxicity. Carfilzomib (CFZ), a novel second-generation irreversible proteasome inhibitor, has demonstrated significant and well-tolerated anti-tumor activity in relapsed/refractory (r/r) myeloma patients. Here we evaluated CFZ's mechanism(s)-of-action and 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 (n=25) were exposed in vitro and/or ex vivo to escalating doses of CFZ or BTZ (0.1-10nM) +/− caspase inhibitors (zVAD-fmk or Q-VD-OPh) for 24, 48 and 72hrs. Changes in ATP content (apoptosis) were determined using the Cell Titer Glow assay and in cell cycle were analyzed by FACScan 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 control versus treated (i.e. CFZ or BTZ) cells to detect PARP-cleavage and changes in Bcl-2 family members or cell cycle regulators. CFZ was found to be 10 times more potent than BTZ and exhibited dose- and time- dependent cytotoxicity against RSCL, RRCL, and primary tumor cells. CFZ has potent anti-tumor activity in tumor cells derived from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL) and other histologies. CFZ exposure induced apoptosis by up-regulation of Bak, Mcl-1 levels and subsequent PARP cleavage in RSCL and (to a lesser degree) RRCL. Co-incubation of RSCL with either BTZ or CFZ and pan-caspase inhibitor led to a significant decrease in BTZ- or CFZ-induced cell death. In contrast, zVAD-fmk and Q-VD-OPh were associated with only partial blocking of BTZ- or CFZ-induced cell death of RRCL. In addition, In vitro exposure of RRCL cells to CFZ (and to a much lesser degree BTZ) reduced RRCL S-phase and induced arrest at G2/M phase. CFZ stabilized G2/M cell cycle regulators cdc2 and cyclinB only in RRCL. Finally, CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumor activity of paclitaxel and vincristine in B-cell lymphoma cell lines and in primary tumor samples. In summary, our data strongly suggest that CFZ is a novel and potent proteasome inhibitor which is has the potential 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. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute. Disclosures: Hernandez-Ilizaliturri: Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy. Czuczman:onyx: Consultancy, Honoraria.

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The role of p21waf1/cip1 and p27Kip1 in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma

Oncogene ◽

10.1038/onc.2013.482 ◽

2013 ◽

Vol 33 (47) ◽

pp. 5415-5423 ◽

Cited By ~ 32

Author(s):

A Newbold ◽

J M Salmon ◽

B P Martin ◽

K Stanley ◽

R W Johnstone

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Tumor Cell ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Tumor Cell Death ◽

Cycle Arrest

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Mitotic catastrophe and cell cycle arrest are alternative cell death pathways executed by bortezomib in rituximab resistant B-cell lymphoma cells

Oncotarget ◽

10.18632/oncotarget.14405 ◽

2016 ◽

Vol 8 (8) ◽

pp. 12741-12753 ◽

Cited By ~ 9

Author(s):

Juan J. Gu ◽

Gregory P. Kaufman ◽

Cory Mavis ◽

Myron S Czuczman ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mitotic Catastrophe ◽

Lymphoma Cells ◽

Cell Death Pathways ◽

Cycle Arrest

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SYK inhibition and response prediction in diffuse large B-cell lymphoma

Blood ◽

10.1182/blood-2011-02-333773 ◽

2011 ◽

Vol 118 (24) ◽

pp. 6342-6352 ◽

Cited By ~ 69

Author(s):

Shuhua Cheng ◽

Greg Coffey ◽

X. Hannah Zhang ◽

Rita Shaknovich ◽

Zibo Song ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Cycle Arrest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Cycle Arrest ◽

Large B Cell Lymphoma ◽

Bcr Signaling ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

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