Abstract 1397: Inhibition of anti-apoptotic Bcl-2 family members leads to synergistic cell death with ER-stress inducers via disruption of autophagy in glioblastoma

Abstract Glioblastoma (GBM) remains one of the most challenging brain tumors, necessitating the exploration of novel therapeutic strategies. The purpose of this study was to employ the inhibitor library and combination screening to identify potential therapies for GBM. We uncovered a model that shows inhibition of anti-apoptotic Bcl-2 family members disrupts the protective autophagy under ER-stress condition, thus aggravating the stress level, which synergizes with the inhibition of Mcl-1 and Bcl-xL to cause enhanced cell death in GBM. Eight GBM cell lines were treated with our in-house library compounds, revealing that a pan-Bcl-2 family inhibitor, obatoclax, effectively reduced cell viability across all GBM cell lines. siRNA-mediated knockdown of anti-apoptotic Bcl-2 family members confirmed that simultaneous targeting of Mcl-1 and Bcl-xL leads to enhanced apoptosis and growth inhibition, demonstrating their roles as potential survival factors in GBM. To further enhance its efficacy, we combined obatoclax with a large chemical library containing over 350 compounds to evaluate potential combination effects. Significant synergistic effects, including reduced IC50 values and enhanced apoptosis induction, were observed between obatoclax and several ER-stress inducers. It was revealed that when GBM cells were undergoing ER stress, inhibition of Mcl-1 and Bcl-xL further exacerbated the stress level and apoptosis, as evidenced by upregulation of ATF-4, CHOP, Bim, and increased caspase-3/7 activity. Such regulation was found to be achieved by the autophagy disruption induced by the presence of obatoclax or the inhibition of anti-apoptotic Bcl-2 family members. The protective autophagy process, serving as a cellular defense mechanism under ER stress, was observed to be terminated in obatoclax-treated GBM cell lines, with decreased autophagic flux, incomplete autophagic degradation, and accumulation of both autophagy markers p62 and LC3-II. Such disruption was proved to directly result in enhanced ER stress and overwhelmed cell homeostasis, leading to significant upregulation of ATF-4, which, on one hand, promotes the translation of pro-apoptotic genes such as CHOP, and, on the other hand, mediates the upregulation of BH3-only proteins like Bim. Bim also cooperates to inhibit anti-apoptotic Bcl-2 family members, especially Mcl-1 and Bcl-xL, contributing to the synergistic cell death in GBM. In conclusion, our study suggests a unique model in which targeting anti-apoptotic Bcl-2 family proteins, such as Mcl-1 and Bcl-xL, coupled with ER-stress induction, demonstrates promising synergism and enhanced cell death in GBM. This highlights the potential of pan-Bcl-2 family inhibitors to overcome current therapeutic limitations in GBM, providing a novel foundation for future therapeutic approaches via the pro-apoptotic targeting method. Citation Format: Tianyi Huang, Satoshi Takagi, Ryohei Katayama. Inhibition of anti-apoptotic Bcl-2 family members leads to synergistic cell death with ER-stress inducers via disruption of autophagy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1397.