Published OnlineFirst May 20, 2016; DOI: 10.1158/1535-7163.MCT-15-0849 Small Molecule Therapeutics Molecular Cancer Therapeutics Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma Daniel Markowitz1, Caitlin Powell1, Nhan L. Tran2, Michael E. Berens2, Timothy C. Ryken3, Magimairajan Vanan4, Lisa Rosen5, Mingzhu He6, Shan Sun6, Marc Symons1, Yousef Al-Abed6, and Rosamaria Ruggieri1 Abstract Medulloblastoma is a cerebellar tumor and the most com- loblastomacellsaswellasUI226patient–derived primary cells, mon pediatric brain malignancy. Radiotherapy is part of the whereas it does not affect the response to radiation of normal standard care for this tumor, but its effectiveness is accompa- brain cells. M443 also inhibits radiation-induced activation nied by significant neurocognitive sequelae due to the delete- of both p38 and Chk2, two proteins that act downstream of rious effects of radiation on the developing brain. We have MRK and are involved in DNA damage–induced cell-cycle previously shown that the protein kinase MRK/ZAK protects arrest.Importantly,inananimal model of medulloblastoma tumor cells from radiation-induced cell death by regulating that employs orthotopic implantation of primary patient– cell-cycle arrest after ionizing radiation. Here, we show that derived UI226 cells in nude mice, M443 in combination with siRNA-mediated MRK depletion sensitizes medulloblastoma radiation achieved a synergistic increase in survival. We primary cells to radiation. We have, therefore, designed and hypothesize that combining radiotherapy with M443 will allow tested a specific small molecule inhibitor of MRK, M443, which us to lower the radiation dose while maintaining therapeutic binds to MRK in an irreversible fashion and inhibits its activity. efficacy, thereby minimizing radiation-induced side effects. We found that M443 strongly radiosensitizes UW228 medul- Mol Cancer Ther; 15(8); 1799–808. Ó2016 AACR. Introduction tion, together with high-dose chemotherapy, has improved 5- year survival rates in patients (2). However, it also causes Radiotherapy is commonly used as part of cancer treat- serious deleterious effects that include neurocognitive deficits, ment, and it is important in the management of 40% of developmental problems, and secondary malignancies in the patients who are cured of cancer (1). The therapeutic benefits majority of survivors (3–6). Identifying new approaches that of radiotherapy are, however, accompanied by late toxicity would allow reduction of the total radiation dose in these that severely affects quality of life, especially in pediatric treatments without compromising therapeutic efficacy is crit- patients. Specifically, in the treatment of medulloblastoma, ical for improving tumor management and quality of life. the most common malignant pediatric brain tumor, radia- Radiosensitizers may offer this opportunity by increasing the efficacy of radiotherapy while reducing effects on the normal developing brain. 1Center of Oncology and Cell Biology, Feinstein Institute, Manhasset, The protein kinase MRK (mixed lineage kinase–related kinase), 2 New York. Translational Genomics Research Institute, Phoenix, Ari- also known as ZAK (sterile alpha motif and leucine zipper con- zona. 3Department of Neurosurgery, Kansas University Medical Cen- ter, Kansas City, Kansas. 4Section of Pediatric Hematology/Oncology/ taining kinase AZK; referred to as MRK in this study; ref. 7), BMT, University of Manitoba, Winnipeg, Canada. 5Biostatistic Unit, belongs to the family of MAPK kinase kinase (MAPKKK) and has 6 Feinstein Institute, Manhasset, New York. Center for Molecular Inno- close homology to the MLK proteins (7–10). MRK has two splice vation, Feinstein Institute, Manhasset, New York. variants, MRKa and MRKb, which share some functions but differ Note: Supplementary data for this article are available at Molecular Cancer in other regards. In particular, while MRKa overexpression pro- Therapeutics Online (http://mct.aacrjournals.org/). motes tumor growth (11), MRKb has been shown to have an D. Markowitz and C. Powell are co-first authors of this article. important role in the response to DNA damage and in the S and M. Symons, Y. Al-Abed, and R. Ruggieri are co-senior authors of this article. G2–M checkpoints (12). At the mRNA level, the two MRK splice b The following authors, Yousef Al- Abed, Marc Symons and Rosamaria Ruggieri, forms are ubiquitously expressed (7, 8), although the MRK have submitted a US patent application: Treatment of solid tumors by inhibiting mRNA is more abundant than the MRKa mRNA. Recently, MRK/ZAK (#50425/507) 2015. This application, however, does not alter the however, the MRKa mRNA has been shown to be abnormally authors' adherence to all the Molecular Cancer Therapeutics policies on sharing spliced in gastric tumors as well as in colorectal, bladder, and data and materials. breast cancers with consequent overexpression of the respective Corresponding Authors: Rosamaria Ruggieri, The Feinstein Institute for Medical protein (13). MRKb is activated by stress (9, 14–16), including Research, 350 Community Drive, Manhasset, NY 11030. Phone: 516-562-3410; ionizing radiation (IR; refs. 7, 12, 17) upon which it contributes to Fax: 519-562-1022; E-mail: [email protected]; and Marc Symons, activation of Chk2 and p38 proteins, which leads to IR-induced [email protected] cell-cycle arrest. MRK silencing caused both failure to arrest cell- doi: 10.1158/1535-7163.MCT-15-0849 cycle progression in response to IR and increased killing by Ó2016 American Association for Cancer Research. radiation (12). www.aacrjournals.org 1799 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 20, 2016; DOI: 10.1158/1535-7163.MCT-15-0849 Markowitz et al. In this study, we examine the role of MRK as a radiosensitiza- Human primary tumor cells and medulloblastoma xenograft tion target in medulloblastoma. In addition, we describe a novel model specific small molecule that we have designed to inhibit MRK The primary UI226 medulloblastoma cells are patient- activity and show that it works as an effective radiosensitizer in an derived xenografts that were established by Dr. Timothy Ryken, orthotopic xenograft model of medulloblastoma. University of Kansas Medical Center (Kansas City, KS). UI226 were largely propagated as flankculturesinnudemiceand Materials and Methods cultured in StemPro media (Life Technologies) for less than 3 weeks before intracranial injections. Cell lines and transfections Allanimalworkwascarriedout in accordance with NIH The human medulloblastoma cell line UW228 (18) was kindly guidelines and was approved by the Institutional Animal Care provided in 2007 by Dr. James Rutka, Hospital for Sick Children, and Use Committee of the Feinstein Institute in Manhasset, NY. Toronto, Canada, and it was authenticated by PCR. Cells were Medulloblastoma cells (5.0 Â 105 UI226 in 5 mLofStemPro cultured in DMEM containing 10% FBS, 1 mmol/L glutamine and medium) were injected over 5 minutes into the cerebellum of 1% penicillin/streptomycin. UMDI cells are a derivative of the 4-week-old athymic female mice (Taconic), using a stereotactic osteosarcoma cell line U2-OS, described in Korkina and collea- frame and the following coordinates: 2 mm to the right and gues (19). In UMDI cells, expressing plasmid pC4-Fv1E-MRK, 1 mm posterior to the lambda, using a sterile dental drill. The MRK can be activated by induced homodimerization with the Hamilton 10-mL syringe loaded with cells was lowered 3 mm drug AP20187 (Clontech). The UMDI cells are cultured in McCoy under the surface of the brain and then lifted for 0.5 mm to media supplemented with 10% FBS, 0.6 mg/mL puromycin, create a pocket for the cells. Three weeks after tumor cell 50 IU/mL penicillin, 50 mg/mL streptomycin, and 2 mmol/L implantation and approximately 2 weeks before the animals L-glutamine. The K-562 and 4T1 cell lines were purchased from became moribund, treatment was started by implantation of an ATCC and cultured according to distributor's protocol. The human osmotic pump (Alzet, Durect) filled with either 0.05 mg/mL neural stem cells (NSC) were purchased from Invitrogen (Thermo solution of M443 or vehicle (0.01% DMSO in PBS) and Fisher Scientific) and cultured in StemPro media (Life Technolo- implanted in a subcutaneous pocket on the dorsal flank of the gies), as directed by the manufacturer. Human astrocytes were animal. A catheter with attached cannula delivered the drug purchased from Gibco (Life Technologies) and cultured in Gibco intracranially and directly into the tumor over a period of 2 astrocyte medium. weeks, at a steady rate of 0.25 mL/hour. Irradiation of the mice The MRK and control luciferase siRNAs sequences were previ- head was initiated 2 days after pump implantation and con- ously described (12) and transfected at a concentration of ducted over 2 days (two fractions of 3 Gy each). It was 10 nmol/L using Dharmafect #1 (Dharmacon) as recommended performed using a biologic irradiator (RS2000, Rad Source by the manufacturer. Technologies), whereas the rest of the body was shielded. A B Figure 1. MRK downregulation radiosensitizes medulloblastoma cells and inhibits downstream signals. UW228 cells were transfected with luciferase control (Luc) or MRK siRNAs, exposed to the indicated doses of radiation and cell survival was determined by the MTT assay (A) or by the clonogenic assay (B) as described in Materials and Methods. The Western blot