Inhibition of anti-apoptotic Bcl-2 family members promotes synergistic cell death with ER stress inducers by disrupting autophagy in glioblastoma
Glioblastoma (GBM) continues to represent one of the most formidable and aggressively challenging brain tumors encountered in clinical practice. Despite considerable efforts in research and the implementation of various therapeutic modalities, current clinical treatment options for this devastating malignancy offer disappointingly limited efficacy, highlighting a critical and urgent necessity for the discovery and development of entirely novel therapeutic strategies. In pursuit of such breakthroughs, our study employed a systematic screening approach, involving both monotherapy and comprehensive combination library screenings. Through this rigorous process, we successfully identified a compelling therapeutic synergy: the efficacy of obatoclax, a pan-Bcl-2 family inhibitor, was significantly amplified when administered in conjunction with various inducers of endoplasmic reticulum (ER) stress, including notably tunicamycin.
Further investigations were undertaken to unravel the molecular underpinnings of this enhanced efficacy. Our findings from combinatorial knockdown experiments targeting key anti-apoptotic proteins provided crucial insights. We observed that the simultaneous reduction of Mcl-1 and Bcl-xL, two prominent members of the anti-apoptotic Bcl-2 protein family, synergistically intensified the induction of apoptosis within GBM cells, particularly under conditions of induced ER stress. This suggests that the combined neutralization of these pro-survival proteins renders glioblastoma cells exquisitely vulnerable to ER stress-mediated death.
Mechanistically, while ER stress inducers alone initiated a cellular stress response in GBM cells, the co-treatment with obatoclax markedly augmented and exacerbated this response. This amplification was evident through the pronounced upregulation of key unfolded protein response (UPR) effectors, specifically activating transcription factor 4 (ATF-4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These proteins are known to drive pro-apoptotic programs under conditions of severe or prolonged ER stress. This enhanced stress response consequently propelled the cells towards apoptosis, as confirmed by a significant increase in caspase 3/7 enzymatic activity and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP), both definitive biochemical markers of apoptotic cell death. Crucially, the functional importance of ATF-4 in this pathway was further established when its genetic knockdown significantly curtailed the apoptosis induced by the combined obatoclax and tunicamycin treatment. This reduction in apoptosis was accompanied by a notable decrease in the expression of both CHOP and BIM, reinforcing ATF-4’s central role in orchestrating the pro-apoptotic signaling cascade.
Moreover, our investigation also delved into the interplay between ER stress and autophagy, a cellular survival mechanism. We observed that under conditions of ER stress, GBM cells typically activate an autophagy response as an adaptive mechanism to recover from the cellular insult. However, the co-treatment with obatoclax critically disrupted these adaptive autophagy responses. Specifically, obatoclax appeared to interfere with the efficient degradation of autophagic cargo, effectively sabotaging the cell’s ability to recycle damaged components and alleviate stress, thereby contributing to the overall cytotoxic effect.
In conclusion, our findings strongly suggest that a dual-pronged therapeutic strategy, specifically targeting the anti-apoptotic proteins Mcl-1 and Bcl-xL in combination with the deliberate induction of ER stress, Foscenvivint could represent a highly promising and innovative approach for the treatment of glioblastoma. This study unequivocally highlights the significant potential of combination therapies involving pan-Bcl-2 family inhibitors to effectively overcome the current limitations and inherent resistance mechanisms that challenge the successful treatment of glioblastoma.