Bortezomib (BTZ) Inhibits p21-Dependent Senescence and Triggers Mitotic Catastrophe in Rituximab-Chemotherapy Resistant Cell Lines (RRCL)

Abstract 1655

The use of rituximab in combination with systemic chemotherapy has not only improved the clinical outcome of patients with B-cell lymphoma, but appears to be changing the biology of relapsed/refractory (rel/ref) disease. Recently, results from the CORAL study described a negative impact from prior rituximab exposure on the responsiveness of diffuse large B-cell lymphoma (DLBCL) to salvage therapy. BTZ, a reversible proteasome inhibitor, has clinically meaningful anti-tumor activity and may overcome chemotherapy resistance in rel/ref lymphomas. We previously demonstrated that the ubiqutin-proteasome system (UPS) plays an important role in the acquirement of rituximab-chemotherapy resistance and that BTZ possesses a caspase-dependent (i.e. BAK stabilization) and a less characterized caspase–independent mechanism-of-action. In our current work, we define the caspase-independent pathways executed following proteasome inhibition in RRCL. Studies were conducted in a panel of rituximab-sensitive (RSCL), RRCL and primary malignant B-cells derived from patients with B-cell non-Hodgkin lymphoma (n = 98). Cells were exposed to BTZ (10nM and 25nM) for up to 48hrs with or without caspase inhibitors. Changes in cell viability and ATP content were determined using the Cell Titer Glo assays. In addition, cell senescence was evaluated by β- galactosidase staining kit. Changes in cell cycle and mitotic index were quantified by flow cytometric analysis. Induction of mitotic catastrophe was detected by confocal microscopy. Changes in key regulatory proteins of apoptosis, cell cycle, autophagy and necrosis were determined by Western blotting. Transient siRNA knockdown of p21, noxa, beclin and other key regulatory proteins were performed in RRCL to evaluate changes in cell cycle arrest, apoptosis, or autophagy and their contribution to the anti-tumor activity of BTZ. In resting conditions, we found that RRCLs proliferate at a slower rate than RRCL. Consistent with this finding was that senescence and cell cycle arrest in S-phase were observed more frequently in RRCL than in RSCL. In vitro exposure of RSCL, RRCL and primary tumor cells derived from lymphoma patients results in both caspase-dependent and -independent cell death. In vitro exposure of RRCL to BTZ led to a reduction in senescence, G2-M phase cell cycle arrest, and subsequent mitotic catastrophe. These changes were not observed in RSCL. Moreover, UPS inhibition by BTZ led to the stabilization of p21, cdc2 and cyclin B in RRCL and in primary tumor cells derived from rel/ref lymphomas. No evidence of autophagy was detected following BTZ drug exposure. Transient knock-down of p21 but not noxa/beclin using siRNA alleviates BTZ induced senescence inhibition, G2-M cell cycle blockage, as well as mitotic catastrophe suggesting that the BTZ-associated anti-tumor effects in RRCL are p21-dependent. Furthermore, in vitro exposure of RRCL to BTZ in combination with M-phase specific chemotherapy agents (e.g. paclitaxel and vincristine) resulted in synergistic activity against RRCL. In summary, our data suggest that bortezomib has a dual mechanism-of-action and is capable to inducing apoptosis or mitotic catastrophe in B-cell NHL cells. We also provide data suggesting that p21 has a pivotal role in response to BTZ in RRCL. A better understanding of the molecular events triggered by BTZ and other UPS inhibitors is necessary to develop novel combination strategies and to develop biomarkers of response that can be utilized to select those patients that are most likely to benefit from this agent. (This work is supported by NIH R01 grant CA136907-01A1).