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effects on caspase-8 cleavage, the combination of both compounds resulted in strong activation of caspase-8 in Jurkat, CCRF-CEM as well as ALL PDX samples (Figure 3C, Online Supplementary Figure S7A,B). Moreover, cleav- age of BAP31 into the pro-apoptotic p20BAP31 fragment or decreased expression of BAP31 proform was only pres- ent in Jurkat, CCRF-CEM and ALL PDX cells treated with the PS89 combination and, as an early trigger, already detectable after 24 h (Figure 3B, Online Supplementary Figure S7). Treatment-independent interaction of procas- pase-8 with BAP31 could be demonstrated by co- immunoprecipitation (Figure 3D). Interestingly, binding of cleaved p43/p41 caspase-8 to BAP31 was only detected in the presence of both stimulants, PS89 and etoposide (Figure 3D, for normalization to BAP31 see Online Supplementary Figure S8). In order to investigate whether the induction of apoptosis by the combination of PS89 and etoposide is critically dependent on caspase-8 activi- ty, cells were stimulated in the presence of the specific and irreversible caspase-8 inhibitor Z-IETD-FMK. As shown in Figure 3E, inhibiting the activity of caspase-8 resulted in diminished apoptosis upon treatment with etoposide and PS89. In accordance, the ratio of apoptotic cells among the cells treated with the PS89 and etoposide combination versus etoposide alone was reduced in cas- pase-8-deficient Jurkat cells compared to wildtype cells (1.7-fold in CASP8-/- versus 2.8 fold in wildtype Jurkat cells (Online Supplementary Figure S9). Next, we examined the functional effects of impairing the expression of the direct PS89 target BAP31 by short interfering (si) RNA. Whereas PS89 significantly enhanced etoposide-triggered apoptosis in control cells, no synergistic effect on apoptosis induc- tion upon treatment with PS89 and etoposide could be detected in siBAP31-transfected cells (Figure 3F).
Endoplasmic reticulum and mitochondrial stress triggers are amplified by PS89
To investigate the consequences of PS89 and etoposide at the ER-mitochondria interface, calcium release from the ER into the cytosol was evaluated by FACS analysis. A shifted Cal 520 fluorescence intensity, indicating higher levels of cytosolic calcium, was observed in Jurkat, CCRF- CEM and HL-60 and ALL PDX cells treated with PS89 in combination than in those treated with etoposide alone (Figure 4A, Online Supplementary Figure S10). Interestingly, amplification of calcium release was observed from 24 h to 48 h in cells exposed to PS89 combination treatment, but not in cells treated with etoposide, daunorubicin or vincristine alone. Loss of mitochondrial membrane integrity, analyzed by JC-1 staining, was increased by co- incubation of etoposide with PS89 (Figure 4B) resulting in release of cytochrome c into the cytosol (Figure 4C, Online Supplementary Figure S11) and elevated levels of reactive oxygen species in Jurkat, CCRF-CEM and HL-60 cells (Figure 4D, Online Supplementary Figure S12). Reactive oxy- gen species signaling from mitochondria to the ER pro- vokes further disturbance of ER redox homeostasis and finally closes the feedback loop.
The eminent role of functional mitochondrial apoptosis signaling was reinforced by studying stable leukemia cells overexpressing the anti-apoptotic proteins Bcl-2 and Bcl- xL. Interestingly, these clones showed a significantly lower sensitivity towards PS89 in combination with etoposide than the empty vector cell line Jurkat/neo (Figure 4E). Moreover, specific targeting of mitochondria with the
PS89 - a novel option for combination therapy in acute leukemia
Bcl-2 inhibitor ABT-199 resulted in synergistic apoptosis in combination with PS89 (Figure 4F, Bliss values are shown in Online Supplementary Table S5), thus substantiat- ing the importance of cytostatic-induced mitochondrial damage provoking the chemosensitizing effect of PS89.
The endoplasmic reticulum-mitochondria interface mediates mutual amplification of PS89 and cytostatic-triggered apoptosis
In summary, PS89 strongly increases mitochondrial apoptosis through a crosstalk and mutual amplification of pro-apoptotic stress signals triggered by cytostatics (Figure 5). The polypharmacological profile of PS89 affecting a network of ER homeostasis proteins is represented by its main targets PDI and BAP31. Upon apoptotic stimuli of cytostatics and exclusively in the presence of PS89, BAP31 is cleaved by caspase-8 to pro-apoptotic p20BAP31. Calcium release from the ER and increased ER stress pro- mote loss of mitochondrial membrane potential (ΔΨm) and apoptosis. In turn, elevated production of reactive oxygen species feeds back to the ER and provokes further ER stress and calcium release. The mutual amplification of ER-mitochondrial stress triggers finally leads to synergistic activation of caspases and apoptosis.
Discussion
In the present study, we demonstrated that activating the apoptotic machinery at the ER-mitochondria interface is a highly promising approach for combination drug treat- ment. Co-stimulation of cytostatics with subtoxic doses of the novel PDI inhibitor PS89 resulted in a highly syner- gistic apoptotic response in a broad range of ALL and AML cell lines and human xenograft cells derived from both newly diagnosed and relapsed patients. In order to exploit this successful strategy, our work examined the intriguing question of how a drug at non-toxic concentra- tions could become highly effective in combination with cytostatics.
The small molecule PS89 was previously identified as a potent chemosensitizing agent, which inhibits PDI.22 Notably, although PDI plays a key role in the maintenance of oxidative protein folding in the ER, no induction of ER stress or unfolded protein response was observed when applying PS89 alone, but only when the molecule was administered in combination with etoposide. This indicat- ed that activation of the ER stress response results from the disability of the ER to resolve a stressful condition which is provoked in cooperation with cytostatics.22
As it is known that many cytostatics induce cell death via activation of the mitochondrial apoptosis pathway34 and, moreover, an increasing number of studies indicate a pivotal role of ER-mitochondria communication in cell fate decisions,35-37 we investigated a potential PS89-trig- gered crosstalk between ER stress and mitochondrial dam- age. Activity-based protein profiling conducted in Jurkat ALL cells identified that, besides PDI, PS89 targets a net- work of proteins located at the ER, including BAP31. Interestingly, it has been described that etoposide stimu- lates caspase-8-mediated cleavage of BAP31 to pro-apop- totic p20BAP31 at the ER-mitochondria interface, which results in calcium release from the ER and induction of mitochondrial apoptosis.14 Moreover, under conditions of ER stress, BAP31 interacts with cell death-inducing p53-
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