ANTICANCER RESEARCH 39 : 3531-3542 (2019) doi:10.21873/anticanres.13499 Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells PATRYCJA WIŃSKA 1, ŁUKASZ WIDŁO 1, KATARZYNA SKIERKA 1, ALICJA KRZYŚKO 1, MIROSŁAWA KORONKIEWICZ 2, JAROSŁAW M. CIEŚLA 3, JOANNA CIEŚLA 1 and MARIA BRETNER 1 1Chair of Drug and Cosmetics Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland; 2Department of Drug Biotechnology and Bioinformatics, National Medicines Institute, Warsaw, Poland; 3Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Abstract. Background/Aim: Recently, we demonstrated the Acute lymphoblastic leukemia (ALL) is the most common ability of inhibitors of protein kinase 2 (casein kinase II; CK2) cancer among children (1), and constitutes approximately to enhance the efficacy of 5-fluorouracil, a thymidylate synthase 25% of cancer diagnoses among children and teenagers (2). (TYMS)-directed drug for anticancer treatment. The present The most common method of treatment of ALL is study aimed to investigate the antileukemic effect of simultaneous chemotherapy, divided into several phases, such as: induction inhibition of dihydrofolate reductase (DHFR), another enzyme phase, consolidation phase, and finally the maintenance involved in the thymidylate biosynthesis cycle, and CK2 in phase, while physicians attempt to reduce the possibility of CCRF-CEM acute lymphoblastic leukemia cells. Materials and relapse (3). Methotrexate, frequently used in the final stage Methods: The influence of combined treatment on apoptosis and of ALL treatment, is a potent inhibitor of dihydrofolate cell-cycle progression, as well as the endocellular level of DHFR reductase (DHFR) (4), a key enzyme in intracellular folate protein and inhibition of CK2 were determined using flow metabolism. After entering the cell, methotrexate undergoes cytometry and western blot analysis, respectively. Real-time polyglutamylation catalyzed by folylpolyglutamate quantitative polymerase chain reaction was used to examine the synthetase. Polyglutamylation causes the retention of influence of silmitasertib (CX-4945), a selective inhibitor of CK2 methotrexate within cells and consequently results in on the expression of DHFR and TYMS genes. Results: The sustained inhibition of DHFR for long intervals (5). Due to synergistic effect was correlated with the increase of annexin V- the fact that in the last stage of treatment, stretching over up binding cell fraction, caspase 3/7 activation and a significant to several years in pediatric patients, the maintenance drugs reduce in the activity of CK2. An increase of DHFR protein level should have the minimal possible side-effects. Unfortunately, was observed in CCRF-CEM cells after CX-4945 treatment, with methotrexate demonstrates potentially serious and life- the mRNA level remaining relatively constant. Conclusion: The threatening toxicities in a subset of patients (6). Therefore, obtained results demonstrate a possibility to improve new therapies are being sought to allow the dose of methotrexate-based anti-leukemia therapy by simultaneous methotrexate to be reduced, these include drug combination inhibition of CK2. The effect of CK2 inhibition on DHFR treatments. It was demonstrated that simultaneous inhibition expression suggests the important regulatory role of CK2- of DHFR and mammalian target of rapamycin kinase had a mediated phosphorylation of DHFR inside cells. synergistic effect on inhibition of leukemia cell proliferation with combination index (CI) values in the range from 0.33 to 0.98 (7). Other studies have shown that the combination of methotrexate and suberoylanilide hydroxamic acid (a This article is freely accessible online. histone deacetylase inhibitor) has a synergistic effect in leukemia cells with combination index values in the range of Correspondence to: Patrycja Wińska, Chair of Drug and Cosmetics 0.64-0.84, depending on the cell line used (for CCRF-CEM Biotechnology, Faculty of Chemistry, Warsaw University of with line: CI=0.84) (8). The most recent research carried out Technology, Noakowskiego St. 3, 00-664, Warsaw, Poland. Tel: +48 222345573, e-mail: [email protected] on L1210 mouse lymphocytic leukemia cell line showed that combined treatment of pretubulysin, a potent tubulin-binding Key Words: Apoptosis, synergism, protein kinase CK2, dihydrofolate antitumoral drug, with methotrexate displayed strong reductase, methotrexate, CX-4945. anticancer effects in both in vitro and in vivo models (9). 3531 ANTICANCER RESEARCH 39 : 3531-3542 (2019) DHFR, with two other enzymes, namely thymidylate and CK2 α were obtained from Cell Signaling Technology (Beverly, synthase (TYMS), and serine hydroxymethyltransferase MA, USA). Monoclonal antibodies against DHFR and (SHMT), constitute the thymidylate synthesis cycle, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from BD Biosciences and Merck Millipore (Darmstadt, providing the substrate required for DNA synthesis and Germany), respectively. Secondary goat anti-rabbit horseradish repair (10). DHFR catalyzes the conversion of dihydrofolate peroxidase-conjugated antibody (IgG-HRP) and anti-mouse IgG to tetrahydrofolate, which is subsequently converted into were purchased from DAKO (Santa Clara, CA, USA). Protease TYMS cofactor, N5,10-methylenetetrahydrofolate, by inhibitors were from Bioshop (Burlington, VT, Canada). High SHMT. Since this cycle is the only de novo source of Capacity cDNA Reverse Transcription Kit was from Applied thymidylate (dTMP), the inhibition of these enzymes induces BioSystems™ (Foster City, CA, USA). Nitrocellulose membrane thymidylate deficiency that affects DNA replication and was from GE Healthcare Life Sciences (Freiburg, Germany), solvents for HRP reaction (Western Bright Peroxide and Western repair, leading to thymine-less death of the cell (11). Here, Bright Quantum) were purchased from Advansta (Menlo Park, CA, we studied the effect of simultaneous inhibition of protein USA). CX-4945 was obtained from Biorbyt. Methotrexate was kinase 2 (casein kinase II; CK2) and DHFR on the viability obtained from Sigma-Aldrich (St. Louis, MO, USA). Other solvents, of leukemia cells, cell-cycle progression, apoptosis and reagents and chemicals were purchased from POCH (Avantor endocellular level of DHFR, CK2 α and phosphorylated Performance Materials, Gliwice, Poland) Merck and Sigma-Aldrich serine 529 of nuclear factor kappa-light-chain-enhancer of Chemical Company (St. Louis, MO, USA). activated B-cells p-65 subunit (NF-ĸB P-Ser529-p65). Cell culture and drug treatment. The aim of our study was the investigation of treatment of CCRF-CEM cell line was purchased from The European Collection of Authenticated Cell Cultures. Cells ALL cells (CCRF-CEM) using methotrexate together with a were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland) selective inhibitor of protein kinase CK2, CX-4945, which supplemented with 20% fetal bovine serum (EuroClone, Siziano, is under stage I/II clinical trial. The efficacy of CX-4945 has Italy) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). been evaluated in a broad range of human hematological Cells were grown in 75 cm 2 cell culture flasks (Sarstedt, Nümbrecht, cancer types, including T-cell ALL (12, 13). Additionally, it Germany), in a humidified atmosphere of 5% CO 2/95% air at 37˚C. was demonstrated that combined treatment with CX-4945 All the experiments were performed in exponentially growing and proteasome inhibitor bortezomib induced synergistic cultures. Stock solutions of the tested compounds were prepared in DMSO and stored at −80˚C for no more than 1 month. Stock apoptotic effects in T- and B- ALL cell lines via reduction of solutions of the tested compounds were diluted 200-fold with the chaperoning activity of heat-shock protein 90 (HSP90) and culture medium to obtain the final concentration of the tested inhibition of NF-ĸB signaling in T-ALL cell lines as well as compounds at 0.5% DMSO. For the combination experiments, stock in primary cells from patients with T-ALL (14). Recently, it solutions of the tested compounds (400-fold) were diluted at a 1:1 was also shown that T-ALL cells express high levels of CK2 ratio with DMSO (when used separately) or with the second compound (when used in a combination). For combination subunits, and that CK2 inhibition by CX-4945 had 4 synergistic effects, promoting the inhibition of survival and experiments CCRF-CEM were seeded at 4×10 cells/well and treated with the compounds, as follows: six to eight concentrations of each interleukin-2 production in T-ALL cells (15). Of note, it was compound or their combination, in the range of 0.125 × drug potency demonstrated that inhibition of CK2 by CX-4945 activated (Dm) to 8 × Dm, i.e. from 0.00275 to 0.176 μM and from 0.3863 to caspase-3 and caspase-7 in cancer cells, with no detectable 24.72 μM for methotrexate and CX-4945, respectively in a constant change of caspase-3/7 activity in normal cells (16). Recently, ratio at two-fold dilution series were used. we demonstrated synergistically induced apoptosis of CCRF- CEM T-ALL cells after simultaneous inhibition of TYMS, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide key enzyme of thymidylate biosynthesis, and protein kinase (MTT)-based viability assay. After incubation with the test CK2 (17). Our previous results showed that combining a compounds, MTT test was performed as