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M. Boudny et al.
discontinuation in a proportion of patients;4 (ii) some patients progress during therapy to the stage of highly adverse diffuse large B-cell lymphoma (Richter transforma- tion);5 and (iii) relapsed/refractory patients harboring 17p deletion (TP53 defect) experience relatively short progres- sion-free survival and overall survival after the single-agent ibrutinib treatment.6,7 Nevertheless, the clinical efficacy of ibrutinib is substantially better compared to chemoim- munotherapy, which has been found to be unsuitable for TP53-defective patients.8
Replication is a vital process for each cancer cell, and the proteins controlling its course represent interesting targets for anti-cancer therapy. The checkpoint kinase 1 (CHK1) supervises replication through the intra-S and G2/M cell cycle checkpoints, where it stabilizes stalled replication forks after DNA damage and participates in DNA repair by homologous recombination process.9,10 CHK1 is an impor- tant member of the DNA damage response (DDR) path- way, which represents a fundamental anti-cancer barrier.11 Central to DDR are two signaling cascades: ATR→CHK1 and ATM→CHK2→p53. While the latter is frequently mutated in tumors, the activity of ATR and CHEK1 genes is essential for cell survival.12,13 In line with this, CHEK1 was found to be an essential gene for 557 out of 558 cancer cell lines, according to the DepMap database (depmap.org) (Online Supplementary Figure S1).
Numerous structurally diverse CHK1 inhibitors have been developed as potentiating agents, i.e. for combination with chemotherapy. Nevertheless, some of them showed interesting pre-clinical single-agent activity against diverse cancer types, including breast and ovarian cancer,14 small- cell lung cancer,15 colorectal cancer,16 neuroblastoma,17 melanoma,18 MYC driven lymphoma,19 and leukemia.20,21 Currently, several CHK1 inhibitors are undergoing evalu- tion in clinical trials focusing on solid tumors and hemato- logic malignancies. In CLL, CHK1 inhibition represents a potentially attractive concept for the following reasons: (i) CHK1 is essential for normal B-cell development and lym- phomagenesis;22 (ii) leukemia and lymphoma cells are par- ticularly vulnerable to CHK1 depletion;20 and (iii) CLL cells are sensitive to manipulation with the level of replication stress (RS), as shown in experiments inhibiting ATR, a CHK1 upstream kinase.23
In our previous study,24 we employed one of the most selective CHK1 inhibitors, SCH900776,25 and showed that it significantly potentiates activity of fludarabine in TP53- mutated CLL cells, as well as in a CLL TP53-wt mouse model. Subsequently, we developed compound MU380, a non-trivial analog of SCH900776 which contains unusual N-trifluoromethylpyrazole moiety protecting the molecule from oxidative dealkylation and thus improving its meta- bolic stability.26 With our current study, we present a robust enantioselective synthesis of MU380, and demonstrate its single-agent efficacy in lymphoid cancer cells. Significantly, to the best of our knowledge, we have for the first time demonstrated the potential for CHK1 inhibition to affect high-risk CLL cells with TP53 defects.
Methods
CHK1 inhibitors
Compound SCH900776 (Merck; MK-8776) was prepared in- house using previously described procedure.24 Compound MU380 was also prepared in-house using our newly developed
enantioselective synthesis (see Results section and Online Supplementary Appendix). These inhibitors were stored at room temperature as 10 mM stock solutions dissolved in DMSO.
Cell lines and primary chronic lymphocytic leukemia cells
Leukemia and lymphoma cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and cultured in accordance with DSMZ recommenda- tions. TP53 mutation status was verified by sequencing, and was in accordance with the International Agency for Research on Cancer database.27 The origin of non-cancerous cell lines is pro- vided in Online Supplementary Appendix. Primary CLL samples consisting of peripheral blood mononuclear cells (PBMNC) with >90% leukemic cells were obtained from patients treated at the Department of Internal Medicine, Hematology and Oncology of the University Hospital Brno. Written informed consent was signed by all patients, and the study was approved by the Ethics Committee of the University Hospital Brno (Project n. 15- 33999A). After thawing, CLL cells were cultured in RPMI-1640 medium with 10% FBS and penicillin/streptomycin. Genetic characterization of the samples is described in Online Supplementary Table S3.
Pro-proliferative stimulation of chronic lymphocytic leukemia cells
Pro-proliferative stimulation of CLL cells was made using the anti-CD40 + IL-4 system developed by Patten et al.28 and used by us previously.24 Here we made the following modifications: the ratio of CLL cells to murine fibroblasts (irradiated by 50 Gy) was 20:1 and length of stimulation was ten days; fresh medium (half volume), and fresh mAb anti-CD40 and IL-4 (full doses of 200 ng/mL and 10 ng/mL, respectively) were added on days 3 and 7. On day 10, CLL cells were gently removed and cultured for an additional three hours (h) to allow residual fibroblasts to attach. For viability testing, CLL cells were transferred to 96-well plates and treated with MU380 or DMSO (mock control).
Transfection of chronic lymphocytic leukemia cells
The cells were transfected by electroporation using Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer´s instructions. Detailed information is provided in the Online Supplementary Appendix.
Cell viability assays, immunoblotting, real-time polymerase chain reaction, analyses of cell cycle, apoptosis and mitotic cells
Detailed information on all methodologies is provided in the Online Supplementary Appendix.
Xenograft experiments
Experiments were approved by the Ethics Committee of the Faculty of Medicine of Masaryk University (n. 47499/2013-8) and performed in accordance with the international ARRIVE guidelines.29 Localized tumors were established in immunodefi- cient NOD-scid IL2Rgnull mice strain30 (Charles River Laboratories, Cologne, Germany) using a subcutaneous injection of MEC-1 cell line (5x106 cells per animal). Mice were matched according to initial tumor size and randomized to treatment with MU380 in 20% aqueous Kolliphor solution (single inhibitor dose 20 mg/kg) or 20% Kolliphor alone. Additional information is included in the Online Supplementary Appendix.
Statistical analyses
Significance level was set as: *P<0.05; **P<0.01; ***P<0.001; = : not significant. The standard level of statistical significance was
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