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.

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