Abstract C45: Comparison of kinetics and mechanism of cell death induction by the proteasome inhibitors bortezomib and marizomib in glioblastoma.

Two proteasome inhibitors, bortezomib (BTZ) and carfilzomib (CFZ), are FDA-approved for treatment of specific liquid tumors, validating the proteasome as a target for cancer therapy. Marizomib (MRZ) is another inhibitor studied in Phase I and II settings. These drugs have different inhibitory profiles that lead to differences in efficacy and toxicity. In leukemia, MRZ causes longer lasting proteasome inhibition and greater cell death than BTZ. These same studies found that MRZ is more dependent on the caspase-8 pathway of apoptosis and production of reactive oxygen species (ROS), while BTZ is more equally dependent on caspases-8 and -9. Given their success in liquid tumors, we were interested in applying these drugs to the solid tumor glioblastoma (GBM), a deadly brain tumor with a median survival time of 15 months. To determine which proteasome inhibitor (PI) was the most potent for GBM, we examined the mechanism of apoptosis induced by BTZ and MRZ as well as the potential for these drugs to cross the blood brain barrier (BBB) to be delivered to brain tumors. We find that BTZ causes longer lasting proteasome inhibition and greater cell death than MRZ in GBM cell lines. This is unexpected, as it is opposite of what was seen in leukemia. Examination of initiating caspase cleavage revealed an early cleavage of caspase-2 (4 h) followed by a later cleavage of caspases-8 and -9 (8-12 h). Early activation of caspase-2 was confirmed using a bimolecular fluorescence complementation model, where caspase-2 induced proximity by recruitment to its activation platforms gives off a fluorescent signal. Furthermore, MRZ induced stronger activation of caspases than BTZ, suggesting that MRZ treatment leads to up-regulation of a resistance mechanism that prevents it from carrying out apoptosis downstream of caspase activation. We also examined the role of ROS in PI-mediated cell death. We treated cells with N-acetyl cysteine (NAC), which increases synthesis of the antioxidant glutathione (GSH). NAC inhibited caspase activation and strongly protected against both BTZ- and MRZ-induced apoptosis. However, raising GSH levels by introducing glutathione ethyl ester did not have the same protective effect. Ongoing studies indicate that NAC is acting independently from its impact on GSH, as NAC still protects cells from proteasome inhibitors when GSH production is inhibited by treatment with buthionine sulfoximine. These data provide insight into mechanisms of PI resistance, perhaps through modulation of the cellular cysteine pool. Though BTZ is more potent in vitro, the question of delivery to brain tumors is an important clinical factor. Using an intracranial, orthotopic model of GBM we find that marizomib causes greater accumulation of the proteasome substrate p27 in brains and tumors of these mice. All together, this research aims to understand the mechanism of these inhibitors to determine the most promising therapeutic regimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C45. Citation Format: Christa A. Manton, Blake Johnson, Lisa Bouchier-Hayes, Joya Chandra. Comparison of kinetics and mechanism of cell death induction by the proteasome inhibitors bortezomib and marizomib in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C45.