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F. Koczian et al.
target protein 1 (CDIP1) leading to cleavage of BAP31, recruitment of Bcl-2 and mitochondrial apoptosis via Bax oligomerization.15 We, therefore, presumed that by inhibiting PDI and further ER stress-related proteins in addition to directly targeting BAP31, PS89 tunes pro-apop- totic feedback from the ER to mitochondria, which results in amplification of cell death signaling in combination therapy.
To confirm the central role of the caspase-8-BAP31 axis in PS89 combination treatment, the processing and acti- vation of the respective proteins was investigated. Indeed, BAP31 cleavage in co-stimulated cells could be observed already at early time points. Interestingly, we were able to show for the first time the intermediate p43/41 cleavage product of caspase-8 associated with BAP31, which still holds a death effector domain. This supports the suggestion that further processing into the finally active p18 fragment does in fact happen at the BAP31 complex.14 As we detected the p43/41 caspase-8 association as well as BAP31 cleavage only in cells exposed to PS89 combination treatment and silencing of BAP31 rescued the chemosensitizing effect of PS89, we conclude that BAP31 binding is crucial for PS89 to medi- ate efficient ER-mitochondria communication. Subsequently, an amplification of calcium release was shown in cells exposed to PS89 combination treatment, but not in those treated with etoposide alone. Ultimately, mitochondria-directed calcium flux promotes mitochon- drial outer membrane permeabilization, accumulation of reactive oxygen species and release of cytochrome c,13,46 which were demonstrated to be increased in PS89 co- stimulated cells as well. Hence, PS89 is able to augment apoptotic triggers of cytostatics by interfering with the ER-mitochondria feedback loop. It is noteworthy that cells stably overexpressing the anti-apoptotic mitochondr- ial proteins Bcl-2 and Bcl-xL are less sensitive to the com- bination treatment, presumably because of an impaired mitochondrial apoptosis machinery. With reference to the concept of the ER-mitochondria ‘social network of cell death’,13 it is conceivable that the ER-mitochondrial feed- back loop procures the crucial pro-apoptotic amplification effect by compromising numerous mitochondria, even if the original stimulus targeted only a few.
In order to further comprehend how stress triggers are communicated from mitochondria to the ER and back, closer examination of the BAP31 complex is required. As shown in previous studies, Fis1 bridges mitochondria and ER-located BAP31 which seems to be further under the control of ER stress-inducible CDIP1 as well as the anti-
apoptotic proteins, Bcl-2 and Bcl-xL.14,15 However, the dynamics regulating the balance of pro- and anti-apoptot- ic proteins within the complex have not been clarified yet. As PS89 is, to our knowledge, the first BAP31-bind- ing small-molecule compound, which facilitates BAP31 cleavage, it might serve as a valuable tool not only for studying the dynamics of the BAP31 protein complex and manipulating decisive BAP31 interactions that favor the pro-apoptotic output, but also for enabling in-depth char- acterization of BAP31 as a prospective pharmacologically addressable target protein in different diseases. With ref- erence to hematologic malignancies, this is further encouraged by the finding that overexpression of BAP31 seems to correlate with chemoresistance, as shown in flu- darabine-resistant mantle cell lymphoma38 as well as pro- teasome inhibitor-adapted myeloma cells.39
In terms of prospective anti-cancer therapies, targeting pro-apoptotic ER-mitochondria crosstalk by combinatory pharmaceutical intervention offers versatile options. For example, BH3 mimetics are a valuable novel class of com- pounds that trigger intrinsic apoptosis and encouraging results were recently shown in an AML phase II trial with ABT-199.40 As shown here, ABT-199 in combination with PS89 strongly increases cell death in Jurkat cells compared to the cell death induced by the agents administered singly. This further underlines the concept of amplifica- tion by communication between ER and mitochondria as a promising strategy for developing new drugs able to trig- ger apoptosis and overcome therapy resistance. Moreover, besides PDI-targeting agents, proteasome or HSP90 inhibitors are potentially highly promising candidates for combination with mitochondria-damaging substances to tune the pro-apoptotic ER stress response.41
In conclusion and in response to the question of how PS89 is able to sensitize acute leukemia cells, the ER-mito- chondria interface was identified as the key platform in the pro-apoptotic signaling cascades mediating the cytotoxic effects of PS89 in combination with cytostatics. By directly affecting PDI and BAP31, PS89 mutually amplifies ER and mitochondrial stress triggers, resulting in strong chemosen- sitizing effects. Hence, this study reveals the potential of targeting the ER-mitochondria apoptosis network as a novel and encouraging strategy in anti-cancer therapy.
Acknowledgments
We thank Judith Hoffmann for the supply of PS89 and the photo probe, and Kerstin Loske as well as Silvia Schnegg for technical assistance. The project was financially supported by the Dr. Robert Pfleger foundation.
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