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This was demonstrated by combinations of PS89 with daunorubicin in HL-60 cells and vincristine in CCRF-CEM cells (Figure 1B, synergism calculations according to the Bliss independence model are shown in Online Supplementary Table S1B) as well as with 6-mercaptopurine and dexamethasone in Jurkat and HL-60 cells, respectively (Online Supplementary Figure S1D-F). Furthermore, vin- cristine-resistant CEM cells showed marked apoptosis in response to treatment with 100 nM vincristine in combi-
PS89 - a novel option for combination therapy in acute leukemia
nation with PS89 (38.3% apoptotic cells), while being resistant to 10-fold higher vincristine concentrations with- out co-stimulation (1000 nM vincristine; 6.8% apoptotic cells) (Figure 1B). Moreover, clonogenic growth of vin- cristine-resistant CEM and HL-60 cells was significantly abrogated upon treatment with PS89 in combination with vincristine or 6-mercaptopurine, respectively (Online Supplementary Figure S1G,H). Following treatment of Jurkat, vincristine-resistant CEM, CCRF-CEM and HL-60
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Figure 2. Analysis of PS89 target proteins. (A) Protein disulfide isomerase (PDI)-genetically modified Jurkat cells (siRNA silencing, 48 h) or HEK cells (PDI overex- pression, 24 h) were treated with etoposide and/or PS89 for 48 h and 72 h, respectively. Apoptosis was determined by FACS analysis and inhibition of proliferation by CellTiter-Blue staining. PDI expression was analyzed by immunoblotting, with actin used as a loading control. (B) Principle of activity-based protein profiling with the PS89 photo probe covalently linked to its cellular targets (adapted from Vomacka et al.30). (C) Volcano plot of target proteins enriched by the PS89 photo probe versus dimethyl sulfoxide (DMSO) (left). Targets with >3-fold enrichment (log2 Probe/DMSO >1.6) and -log10 P-value >2 are highlighted. Ranking of targets according to the degree of competition by unmodified PS89 (right). (D,E) Target network analysis of the 42 most enriched PS89-binding proteins with STRING v10. Proteins involved in the most prominent gene ontology classes are highlighted (Cellular Component - green circles; Biological Process - green dots).
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