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specific networks and for overcoming the system robust- ness of cancer cell phenotypes.10,11 Translating this concept to combinatorial drug treatment, a highly interesting issue is not only how networks are locally perturbed by individ- ual compounds, but moreover how interventions at mul- tiple cellular loci cooperate. Considering potential pro- apoptotic target networks, the crucial role of the endoplas- mic reticulum (ER)-mitochondria ‘social network of cell death’ was recently stressed in several studies highlighting the dynamic interaction of these two cellular elements.12,13 In this context, the B-cell receptor-associated protein 31 (BAP31) was described as a substrate of caspase-8 and emerges as a communicator of apoptosis signals from the ER to mitochondria.14,15 Consistently, a role of the caspase 8-BAP31 axis has been demonstrated in ER stress-trig- gered apoptosis of B-cell lymphocytic leukemia cells.16 ER stress results from an imbalance between ER protein load and folding capacity. Protein disulfide isomerases (PDI) constitute a crucial family of enzymes for maintaining oxidative protein folding and ER homeostasis.17 Hence, these proteins have been recognized as exciting novel tar- gets in cancer research.18 Furthermore, overexpression of PDI has been discovered in leukemia and linked to chemoresistance.19-21
Recently, we introduced the first reversible small-mole- cule PDI inhibitor PS89 which binds in close proximity to the catalytic centers of PDI.22 Moreover and contrary to other PDI inhibitors that are severely cytotoxic,23,24 PS89 is not toxic up to micromolar concentrations, although it has been shown to greatly enhance etoposide-induced apop- tosis. This exceptional feature of effective chemosensitiza- tion at subtoxic doses motivated not only further combi- nation therapy studies with PS89, but also a deeper analy- sis of its interactive signaling. In the present work, PS89 is set on stage as a novel therapeutic option for the treat- ment of acute leukemia. The favorable attributes of PS89 and its broad applicability are highlighted in ALL and AML cell lines, drug-resistant cells as well as patient- derived xenograft (PDX) cells. The critical networks inte- grated in the synergistic pro-apoptotic signaling of PS89 in combination with cytostatics were identified, thus emphasizing the crucial function of ER-mitochondria communication for successful combination therapies.
Methods
Cell cultures
Jurkat cells (wild-type, CASP8-deficient, Bcl-2- and Bcl-xL-over- expressing) were kindly provided by P. H. Krammer (Heidelberg, Germany). CCRF-CEM and vincristine-resistant CEM cells25,26 were obtained from M. Kavallaris (Sydney, Australia), HEK 293 and HeLa cells from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and HL-60 from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were maintained in ATCC-recommended culture conditions.
Patient-derived xenograft cells, peripheral blood mononuclear cells and CD34-positive cells
The model of ALL and AML patients’ leukemia cells growing in mice has been described previously.27,28 Ethical statements and approvals are outlined in the Online Supplementary Information. In the present study, PDX cells were freshly isolated from the bone marrow or spleen of NSG mice and cultured in the presence or
PS89 - a novel option for combination therapy in acute leukemia
absence of compounds. Peripheral blood mononuclear cells were freshly isolated from EDTA-anticoagulated blood of healthy donors by gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) according to the man- ufacturer's instructions. The peripheral blood mononuclear cells were maintained in RPMI 1640 with 2 mM glutamine supple- mented with 20% (v/v) fetal calf serum and 1 mM pyruvate. CD34-positive cells were identified by staining with fluorescein isothiocyanate-conjugated anti-human CD34 antibody (BD Biosciences, Heidelberg, Germany) according to the manufactur- er’s instructions and analyzed by flow cytometry as described by Fukuda et al.29
Ethical statements
Written informed consent was obtained from all patients or legal guardians when the patients were minors. The study was performed in accordance with the ethical standards of the respon- sible committee on human experimentation (written approval from the ethics committee of the Ludwig Maximilian University Hospital, Munich, number 068-08) and with the Helsinki Declaration of 1975, as revised in 2000. All animal experiments were performed in accordance with the current ethical standards of the official committee on animal experimentation (written approval from Regierung von Oberbayern, number 55.2-1-54- 2532-95-10).
haematologica | 2019; 104(3)
Activity-based protein profiling
Jurkat cells were incubated with unmodified PS89 (100 mM) or dimethyl sulfoxide (DMSO) as a control for 45 min at 37°C and in a second step with the PS89 photo probe (20 mM) or DMSO as a control for 45 min at 37°C. Cells were lysed in 1 mL phosphate- buffered saline with 1% (v/v) NP40 and 1% (w/v) sodium deoxy- cholate and sonication for 15 s on ice. Sample preparation and mass spectrometry analysis of target proteins by gel-free activity- based protein profiling and dimethyl labeling was performed as previously described.30 Cutoff criteria for target identification were: (i) enrichment by photo probe log2 Probe/DMSO >1.6, -log10 P-value >2 and (ii) PS89 competition log2 Probe/PS89 >0. The data shown are the results of three biological replicates.
Details of the methods used are available in the Online SupplementaryAppendix.
Results
PS89 sensitizes acute leukemia cell lines and patient- derived xenograft cells to cytostatics
The concept of chemosensitization with the recently introduced PDI inhibitor PS89 was initially evaluated in a dose-response apoptosis assay. While PS89 was applied at a fixed subtoxic dose, the concentration of etoposide could be reduced by at least half to achieve equal cytotox- icity applying the combination treatment. This is in line with a more than 2-fold shift in the half maximal effective concentration (EC50) (Figure 1A). Bliss values indicating synergistic effects of PS89 in combination with etoposide are shown in Online Supplementary Table S1A. Whereas etoposide-treated cells showed a pronounced G2 arrest, PS89 had no effect on the cell cycle (Online Supplementary Figure S1A). PS89 treatment in combination with subtoxic concentrations of etoposide synergistically inhibited Jurkat cell proliferation and colony formation (Online Supplementary Figure S1B,C). The ability of PS89 to induce synergistic apoptosis with diverse cytostatics could be translated to acute leukemia cells of different lineages.
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