Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma.

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.


Supplementary
: Analysis of SK053 binding to PRDX1 structure. A. Superposition of PRDX1 structures in various stages of catalysis: reduced form (PDB code 2Z9S, green), oxidized form (PDB code 1QQ2, blue) and in the complex with sulfiredoxin (PDB code 3HY2, yellow). Active site Cys residues are displayed as red sticks and labeled. Variable positions of Cys173 are indicated with arrows. B. Docking of SK053 molecule to the binding pocket around Cys52 of oxidized PRDX1 (PDB code 1QQ2), in which all ratspecific residues were substituted with human equivalents. Left panel: Subunits A and B of PRDX1 dimer are displayed in blue and green color, respectively. Active site Cys residues are displayed as red sticks and labeled. Distance between ligand and cysteines is indicated. Right panel: Model of PRD1 dimer, in the same orientation, shown in the surface representation, colored according to the distribution of the electrostatic surface potential calculated with Adaptive Poisson-Boltzmann Solver program, blue-positively charged regions, rednegatively charged regions. Graphics are prepared with PyMOL. Raji cells were treated with SK053 for the indicated times and the levels of ROS were detected with CellROX green fluorescent probe (ThermoFisher), according to manufacturer's protocol. The green fluorescence intensity was evaluated by flow cytometry, and the ROS levels are presented as the fold change over untreated control. The mean values from two independent repeats ± SD are shown, *P < 0.05. Figure S10: Detection of SK-bio-binding proteins in Raji cell lysates by immunoblotting. Raji-sub lysates were incubated with SK-bio or SK-in, and biotin-binding proteins were isolated by neutravidin-affinity pull-down. Subsequently, the proteins eluted from beads were separated by SDS-PAGE and subjected to immunoblotting. The results indicate a pronounced capacity of the genes to differentiate the studied functional classes of B cells.

Chemical synthesis of biotin-labeled SK053 (SK-bio) and its inactive counterpart (SK-in)
Biotinylated compound SK-bio was obtained from corresponding alcohol 2. Compound 3 was prepared according to the method applied for SK053 [1]. The compound 2 was prepared as follows: the solution of compound 3 (58 mg, 0.1 mmol) in methanol (1.2 ml) was cooled to 0 °C. Then, the aqueous solution of potassium carbonate was added (2 M, 64 μl). The progress of the reaction was monitored by TLC. After 15 min, methanol was evaporated in vacuo without heating. Dichloromethane (10 ml) and the brine (3 ml) were added. Then, the aqueous phase was extracted with dichloromethane (3 × 5 ml). The organic phase was dried over magnesium sulfate, filtered and evaporated. The colorless oil can be used without further purification (51 mg, 95% yield).
Compound 2 (SK-bio) (24 mg, 0.05 mmol) was dissolved in DMF (1 ml). Then D-biotin (13 mg, 0.05 mmol), DMAP (cat. 2 mg) were added. The solution was cooled to 0 °C and EDC.HCl (10 mg, 0.05 mmol) was added. The reaction was carried out for 28 hours at RT. Then DMF was removed in vacuo. Crude product was purified by preparative TLC plates (silica gel, chloroform:methanol 10:1). 12 mg of the biotinylated compound SK231 was obtained as a diastereoisomeric mixture (1:1) with 34% yield. 1   Compound 5 (SK-in) was obtained from L-Leucine as described before [2]. Compound 6 was prepared as follows: To a solution of compound 5 (290 mg, 1.1 mmol) in toluene (2 ml) the ethanolamine was added (605 mg, 10 mmol). The reaction was carried out at 50 °C for 24 h. Toluene was removed in vacuo and the crude product was recrystallized from hexane:ethyl acetate. The 206 mg of white solid was obtained (64% yield). Product 6 (100 mg, 0.34 mmol) was then dissolved in DMF. DMAP (13 mg) and D-biotin (92 mg, 0.37 mmol) were added. Then the reaction was cooled to 0 °C and EDC.HCl was added (72 mg, 0.37 mmol). After 24 h DMF was removed in vacuo. The crude product was dissolved in chloroform and the organic layer was washed with citric acid (10%, aq.) and sodium bicarbonate (8%, aq.). The organic layer was washed with brine and evaporated. The product was purified using preparative TLC plates (silica gel, ethyl acetate). 68 mg of colorless oil was obtained (38%) 1

Protein expression and purification
Bacteria were grown in LB medium at 37 °C and protein expression was induced at an OD600 of 0.7-0.9 with 1 mM IPTG (isopropyl β-D-1thiogalactopyranoside, Fluka) and continued overnight. Bacterial cells were collected by centrifugation, the pellet was resuspended in 10 ml of lysis buffer consisting of CelLytic B (Sigma-Aldrich), 0.2 mg/ml lysozyme, 50 U/ml benzonase, and lysed for 15 min at RT. Cell debris was removed by centrifugation and the supernatant loaded onto the HiTrap FF crude 5-ml Ni Sepharose column (GE Healthcare Life Sciences AB) equilibrated with binding buffer: 20 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol. The protein was eluted using 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol buffer with imidazole gradient varying from 10 to 400 mM. The protein was further purified with gel filtration chromatography using Superdex75 10/300 GL column (GE Healthcare Life Sciences) equilibrated with 10 mM Tris 7.5, 100 mM NaCl, 10 mM DTT buffer. The nickel affinity chromatography and subsequent gel filtration were performed using Akta Avant 25 operated by Unicorn 6 software (GE Healthcare Life Sciences). The eluted fractions containing PRDX1 were concentrated using VivaSpin 10 kDa concentrator (Sartorius), mixed 1:1 with glycerol and stored at −20°C.

PRDX1 activity assay
The activity of PRDX1 was measured in a NADPHdependent assay, based on [3]. PRDX1, pre-reduced with TRX-TRXR, was incubated with different concentrations of SK053 for 1 h and dialyzed against reaction buffer to remove unbound inhibitor and exclude the possibility of TRX/TRXR inhibition, and the reaction was initiated with the addition of TRX, TRXR and NADPH. The activity was measured in a final volume of 100 μL containing the following: reaction buffer, 2.8 μM TRX (IMCO), 0.1 μM TRXR (IMCO), 1 mM H 2 O 2 , 150 μM NADPH and 10 μM PRDX1. Oxidation of NADPH was measured as a change in the absorbance at 340 nm (Asys UV340M spectrophotometer). Data points were measured in triplicate in individual experiments, and error bars represent the SD. The IC 50 values were evaluated from two independent experiments, with Four Parameter Logistic Standard Curves Analysis, using SigmaPlot software.

In vitro binding experiments
Prior to treatment with the studied compounds, purified PRDX1 was dialysed against buffer containing 25 mM potassium phosphate pH 7.0, 1 mM EDTA, 100 mM (NH 4 ) 2 SO 4 . Upon dialysis, PRDX1 at a concentration of 0.1 μg/μl was incubated with 20 μM SK-bio or SK-in in the dialysis buffer at 37 °C at the indicated times.