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.
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.
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.
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