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 ◽  
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.

Obatoclax and Bortezomib Therapy Results in Disruption of the p53-Mediated Apoptosis/Autophagy Pathway and Is Associated with Potent Synergistic Anti-Tumor Activity against Rituximab-Chemotherapy-Sensitive and -Resistant B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 288-288 ◽  
Author(s):  
Francisco Hernandez-Ilizaliturri ◽  
Ping-Chiao Tsio ◽  
Ryan Campagna ◽  
Cory Marvis ◽  
Wasif Riaz ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Synergistic Activity ◽  
Knock Down ◽  
Anti Tumor Activity

Abstract Abstract 288 Interaction between the members of the BH3 domain family of proteins plays an important role in the development, progression, and prognosis of various subtypes of B-cell lymphoma. Therapies that selectively induce a pro-apoptotic environment are an attractive strategy to overcome chemotherapy-resistance in B-cell lymphoma. The proteasome is an important regulator of various members of Bcl-2 family proteins. We previously demonstrated that obatoclax, a novel BH3 mimetic, was able to enhance the anti-tumor activity of rituximab and chemotherapy agents and induced both apoptosis and autophagy in B-cell lymphoma. In an attempt to increase the therapeutic options for B-cell lymphoma patients we studied the biological effects of obatoclax in combination with bortezomib in a panel of rituximab-[chemotherapy]-sensitive (RSCL) and rituximab-[chemotherapy]-resistant cell lines (RRCL), as well as primary tumor cells isolated from 45 NHL patient biopsy samples with various subtypes of B-cell lymphoma: (ie including, DLBCL, follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and Hodgkin lymphoma (HL)]. Patient-derived primary tumor cells were isolated from fresh biopsies by negative selection using magnetic beads. Transient knock-down of p53 and Noxa were performed to determine the role of p53 in the anti-tumor activity of bortezomib or obatoclax, respectively. NHL cells were exposed in vitro to escalating doses of obataclox (0, 2, 5, 10 and 20mM) and/or bortezomib (0, 2, 10 and 20nM) for 24 and 48 hrs. Cell death was determined by the cell glow luminescent assay and DNA synthesis was evaluated by standard [3H]-Thymidine incorporation assays at 24 and 48 hrs. Changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. In vitro exposure of RRCL, RSCL, and primary tumor cells to the combination of obatoclax plus bortezomib demonstrated significant synergistic activity compared to controls. Patients with DLBCL (n=15) and FL (N=12) demonstrated significant sensitivity to this combination. Of note, activity was observed in patients with either de novo or relapsed/refractory germinal B-cell (GBC) or activated B-cell (ABC) DLBCL (as characterized by the Han's criteria). Additionally, cell death induced by obatoclax or bortezomib could be inhibited by transient knock-down of p53 or Noxa, respectively. In summary, deregulation of apoptosis by BH3 inhibition with obatoclax and bortezomib results in cell death and antiproliferation not only in RSCL and RRCL, but also in primary tumor cells derived from “treatment-naïve or refractory” DLBCL and FL patients. Data strongly suggests that both p53 and Noxa have pivotal roles in response to obatoclax and bortezomib. The combination of obatoclax plus bortezomib has the potential of becoming a novel and potent therapeutic strategy in the treatment of B-cell lymphoma in the future. 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.

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.

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.

Apoptotic and Non-Apoptotic Cellular Pathways Executed by Bortezomib (BTZ) in Rituximab-Resistant Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4967-4967
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
Karen E Thudium ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mitotic Catastrophe ◽  
M Cell ◽  
Lymphoma Cells

Abstract Abstract 4967 Rituximab-chemotherapy relapsed/refractory (r/r) B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. A better understanding of the mechanism(s)-of-action of BTZ and other proteasome inhibitors (PI) is likely to aid in the identification of biomarkers that can be used to determine clinical responsiveness and/or help in the rational development of novel PI-based therapeutic combinations (e.g. incorporating biologics, small molecules and/or chemotherapy) in r/r B-cell lymphoma. Previously we demonstrated that rituximab resistance was associated with increased proteasome activity leading to a de-regulation in the apoptotic threshold of lymphoma cells to multiple chemotherapy agents. Pharmacological and genetic (e.g. siRNA silencing of BAK/BAX) inhibition of apoptosis partially affected BTZ activity in rituximab-resistant (RSCL) but not in rituximab-sensitive cell lines (RSCL) suggesting the existence of alternative pathways of cell death associated with PI exposure. To this end we evaluated the contribution of cellular senescence, cell cycle inhibition, or mitotic catastrophe to the anti-tumor activity of BTZ as a single agent or in combination with chemotherapeutic agents in RSCL, RRCL and in primary tumor cells. Lymphoma cells were exposed to BTZ (10-25nM) for 24–48 hrs. Cell senescence was determined by SA-β-gal staining using a senescence assay kit and inverted phase-contrast microscopy was performed. Changes in cell cycle were analyzed by the FACScan DNA method and changes in cell cycle regulatory proteins (i.e. cdc2, cyclinA/B, p21, CDK2/4/6) were analyzed by Western blotting. Mitotic index was determined by Wright-Giemsa stain and positive cells were counted under a Nikon microscope. Mitotic catastrophe was determined by confocal microscopy by staining with α-tubulin antibody. Finally, changes in ATP content was determined by the Cell Titer Glo assay. Baseline differences were observed between RSCL and RRCL in terms of cell morphology, proliferation rate and senescence. RRCL (Raji2R and Raji4RH) were considerably larger in size, had a slower proliferation rate and an exhibited a 3-fold increase the number of cells in senescence than RSCL. In vitro exposure of RSCL and RRCL to BTZ attenuated the number of cells in senescence by 50–75%. Cell cycle analysis demonstrated that RRCL had more cells in S phase when compared to RSCL. In vitro exposure to BTZ-induced G2/M arrest in RRCL, but not in RSCL. Overexpression of G2/M cell cycle regulatory proteins cyclin B and cdc2 were observed in RRCL and in tumor cells isolated from r/r B-cell lymphoma patients. Mitotic catastrophe with multi-nucleated cells were only detected in RRCLs exposed to BTZ. In vitro and ex vivo exposure of RSCL and RRCL to BTZ potentiated the cytotoxic effects of paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL and primary tumor cells isolated from r/r lymphoma patients in a dose-dependent manner. Our results suggested that BTZ activates several death pathways in B-cell lymphoma pre-clinical models. In addition to apoptosis, BTZ is capable in triggering mitotic catastrophe in rituximab-chemotherapy lymphoma cells with decreased levels of pro-apoptotic proteins. Moreover, sensitization of RRCL to drug therapy involves interplay between cellular senescence attenuation, G2/M cell cycle regulation, and mitotic catastrophe. Hence, proteasome inhibition may provide a novel therapeutic approach for treating apoptosis-resistant 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:Millennium: Honoraria, Research Funding.

Entinostat (SNDX-275), a Novel DAC Inhibitor, Is Highly Effective in Rituximab- [Chemotherapy]-Sensitive or Rituximab-[Chemotherapy]-Resistant Lymphomas and Has Synergistic Anti-Tumor Activity When Combined with Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3727-3727 ◽  
Author(s):  
Umeer Ashraf ◽  
Myron S. Czuczman ◽  
Cory Marvis ◽  
John Gibbs ◽  
Francisco J Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
Synergistic Activity ◽  
Kinetic Assay ◽  
Anti Tumor Activity

Abstract Abstract 3727 Poster Board III-663 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Entinostat (SNDX-275) is a novel and potent DAC inhibitor that is selective for class I DACs and is currently undergoing pre-clinical and clinical testing in Hodgkin lymphoma (HL). Potent synergistic anti-tumor activity has been observed by combining less potent DAC inhibitors with bortezomib in pre-clinical models. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphoma, we evaluated the effects of Eentinostat as a single agent and in combination with bortezomib against B-cell non-Hodgkin's lymphoma (NHL) cell lines and primary NHL cells. Studies were conducted in a panel of 12 NHL cell lines representing various subtypes of B-cell lymphoma (i.e. DLBCL/ABC, DLBCL/GCB, Burkitt's, transformed and MCL), which included: rituximab-[chemotherapy]-sensitive cell lines (RSCL, Raji, RL and DHL-4), rituximab-[chemotherapy]-resistant cell lines (RRCL, Raji-4RH, Raji-2R, RL-4RH, and DHL-4 4RH), and primary lymphoma cells isolated from patients with various subtypes of NHL and HL. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells and patient-derived primary cells were exposed to entinostat at different doses (0.01 to 100uM) either alone or in combination with CDDP (1 to 100μM), doxorubicin (4 to 16μM), vincristine (1 to 5μM), or bortezomib (1 to 10nM). Anti-tumor activity was measured after a 24 or 48 hr incubation. In cell lines, changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. For patient-derived primary NHL cells, changes in ATP content (apoptosis) was determined using the cell titer glow assay. Entinostat was highly active in all the cell lines tested including rituximab-[chemotherapy]-resistant cell lines. The IC50 of Entinostat in the majority of the cells tested was 0.5 to 5uM at 48 hrs. Similar findings were observed in primary tumor cells derived from lymphoma patients. In addition, synergistic activity was observed by combining entinostat and bortezomib in both NHL cell lines, as well as in primary NHL/HL tumor specimens. A lesser degree of augmented anti-tumor activity was also observed when entinostat was combined with cisplatin or doxorubicin (but not vincristine). In summary, our data suggests that entinostat is a novel and potent DAC inhibitor with a wide therapeutic spectrum. Entinostat is capable of inducing cell death against various subtypes of B-cell lymphoma cell lines including RSCL, RRCL, as well as patient-derived primary tumor cells and augments the anti-tumor effects of bortezomib and other chemotherapeutic agents. Given the isoform selectivity of entinostat, the results indicate that HDAC1 and 2 may be the key targets of DAC inhibitors in HL and NHL cells. Ongoing studies are evaluating the mechanisms responsible for the synergistic effects of entinostat plus chemotherapy and will be updated at the annual meeting. Current findings strongly suggest that entinostat added to bortezomib and/or other chemo agents may become a novel and potent strategy in the treatment of aggressive and indolent NHL and HL in the future. 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.

Dual Inhibition of PKC Delta By Carfilzomib (CFZ) and Ibrutinib Results in Synergistic Activity in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models and in Primary Tumor Cells Isolated from B-Cell Lymphoma Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4006-4006
Author(s):  
Qunling Zhang ◽  
Juan J Gu ◽  
Ye Guo ◽  
Jacob Klos ◽  
Cory Mavis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Caspase 3 ◽  
Cell Lymphoma ◽  
Biological Effects ◽  
Active Form ◽  
B Cell Lymphoma ◽  
Mitochondrial Potential

Abstract Introduction: A better understanding in the role of the B-cell receptor-signaling pathway in lymphomagenesis led to the clinical development of ibrutinib is an orally bioavailable Bruton's tyrosine kinase inhibitor (BTKi). Ibrutinib has clinical activity in several subtypes of B-cell lymphoma (follicular lymphoma, mantle cell lymphoma and certain diffuse large B-cell lymphoma [DLBCL] subtypes). Recently, an increasing number of ibrutinib-treated patients are developing disease progression, stressing the need to optimize its therapeutic potential. To this end, we evaluated the biological effects and interactions of ibrutinib and CFZ, a non-reversible proteasome inhibitor in several lymphoma pre-clinical models including primary tumor cells isolated from lymphoma patients. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines as well as primary tumor cells isolated from B-cell lymphoma patients (N=20) were exposed to ibrutinib and/or CFZ. Changes in cell viability were evaluated by Presto Blue and Titer Glo assays respectively. Coefficient of synergy was determined using the CalcuSyn software. To further study the mechanisms responsible for the biological effects of ibrutinib and CFZ, RSCL and RRCL were exposed to Ibrutinib+/- CFZ for 48hr. Changes in apoptosis, mitochondrial potential, and cell cycle distribution were evaluated by flow cytometry by AnnexinV-PI, DiOC6 or PI staining respectively. Subsequently, protein lysates were isolated from ibrutinib+/- CFZ exposed RSCL or RRCL and changes in downstream targets (PKCδ a member of diacylglycerol-dependent, calcium-independent serine/threonine-specific protein kinases PKC isoform know to be a substrate of caspase 3 and able to mediate apoptosis), Bcl-2 family members and cell cycle regulatory proteins were evaluated by Western blotting. Results: In vitro exposure of RSCL, RRCL, and primary tumor cells to ibrutinib and CFZ resulted in synergistic anti-tumor activity. In addition, the combination of ibrutinib and CFZ resulted in a more pronounced decrease in the mitochondrial potential, higher degree of apoptosis (as demonstrated by flow cytometry and caspase 3 or PARP cleavage) and increase in G1 cell cycle arrest when compared to single agent ibrutinib or CFZ. Perhaps related to these findings, we found that exposure of lymphoma cells to Ibrutinib and CFZ resulted in an increase in caspase 3, the active form of PKCδ and BAX levels. Using caspase inhibitor Q-VD-OPh rescued the combination killing effect of ibrutinib and CFZ, as well as abrogated induction of the active form of PKCδ. In addition, Ibrutinib in combination with CFZ decreased cyclin E and CDK2 levels. Conclusions: Together our data suggests that ibrutinib and CFZ had a strong synergistic anti-tumor activity against rituximab-chemotherapy sensitive and resistant lymphoma cells and patient samples. At the molecular level, the combination of ibrutinib and CFZ affects the balance of the apoptotic machinery and key regulatory proteins of the cell cycle that could play an important role in the synergistic effects observed. Further signaling and in vivo studies are necessary to evaluate the therapeutic role of ibrutinib in combination with carfilzomib in 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 Funds). Disclosures Czuczman: MorphoSys: Consultancy; Celgene: Employment; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.

Molecular Inhibition of mTOR with Temsirolimus (TORISEL™, CCI-779) Is a Promising Strategy in Relapsed NHL: The University of Chicago Phase II Consortium.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2483-2483 ◽  
Author(s):  
Sonali M. Smith ◽  
Barbara Pro ◽  
Scott Smith ◽  
Patrick Stiff ◽  
Eric Lester ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Phase Ii ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mtor Inhibition ◽  
Promising Strategy ◽  
Biomarker Analysis ◽  
Grade 3

Abstract Mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase positioned at the juncture of several signal transduction pathways frequently disrupted in cancer. Downstream effectors, S6KI and 4EBP1, regulate cell cycle kinetics and cell growth via translational modulation, and this is effectively halted by rapamycin. Temsirolimus (TORISEL™, CCI-779) is a rapamycin ester derivative with cell cycle inhibitory properties. We conducted a phase II study of temsirolimus 25 mg IV over 30 minutes weekly in three cohorts of patients with relapsed/refractory NHL: Group A (diffuse large B-cell lymphoma, DLCL), Group B (follicular lymphoma, FL), Group C (small lymphocytic lymphoma/chronic lymphocytic leukemia [SLL/CLL] and other indolent B-cell lymphomas). Correlative studies in primary tumor tissue and in peripheral blood samples include phosphorylated p70S6K, total and phosphorylated 4EBP1, PTEN status, p21 status, and p27 status. Forty patients are registered, of which 21 are evaluable for response. Six patients are inevaluable and 13 too early for response evaluation; all patients were included in toxicity evaluation. There were 22 males/18 females with a median age of 59 (range, 29–83). Primary tumor samples were collected in 10 patients, with biomarker analysis pending. Histologic subtypes include 7 DLCL, 14 FL, and 13 CLL/SLL. Of 21 evaluable patients for response, there were 3 complete remissions (all FL) and 5 partial remissions (1 DLCL, 3 FL, 1 SLL), for an overall response rate of 40%. An additional 10 patients (5 FL, 6 CLL/SLL) had minor responses or stable disease; only 3 pts had progressive disease as their best response. The median follow-up is 4 months (range, 1–15 months). Grade 1–2 non-hematologic toxicities (except where noted) include increased hepatic enzymes (10 pts), skin rash (13 pts), fatigue (18 pts; grade 3 in 2 pts), hypercholesterolemia (22 pts), hyperglycemia (21 pts; grade 3 in 1 pt), hypertriglyceridemia (20 pts; grade 3 in 1 pt). Six patients were removed from study due to pneumonitis; 4 of these patients were symptomatic (cough, fevers, dyspnea) and 2 were asymptomatic but had pulmonary abnormalities on staging CT scans. Grade 3 and 4 hematologic toxicities include thrombocytopenia (5 pts), lymphopenia (5 pts), and leucopenia (2 pts). In summary, temsirolimus shows preliminary but encouraging activity across multiple B-NHL subtypes, complementing the published activity in mantle cell lymphoma. Careful attention must be paid to pulmonary toxicity. However, the early activity, including disease regression and stabilization, suggests that future combinations with cytotoxics or other biologics may be warranted, and that mTOR inhibition in NHL is a promising strategy.

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

Blood ◽  
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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