MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma Jonathan B

Published OnlineFirst June 30, 2016; DOI: 10.1158/1541-7786.MCR-16-0172

Signal Transduction Molecular Cancer Research MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma Jonathan B. Bell1, Frank D. Eckerdt1,2, Kristen Alley1, Lisa P. Magnusson1,2, Hridi Hussain1,2, Yingtao Bi3, Ahmet Dirim Arslan1,4, Jessica Clymer1,5, Angel A. Alvarez1, Stewart Goldman1,5, Shi-Yuan Cheng1, Ichiro Nakano6, Craig Horbinski1,2,7, Ramana V. Davuluri1,3, C. David James1,2, and Leonidas C. Platanias1,4,8

Abstract

Glioblastoma multiforme remains the deadliest malignant a crucial effector for MNK-induced mRNA translation in cancer brain tumor, with glioma stem cells (GSC) contributing to treat- cells and a marker of transformation. Importantly, merestinib ment resistance and tumor recurrence. We have identiﬁed MAPK- inhibited growth of GSCs grown as neurospheres as determined interacting kinases (MNK) as potential targets for the GSC pop- by extreme limiting dilution analysis. When the effects of mer- ulation in glioblastoma multiforme. Isoform-level subtyping estinib were assessed in vivo using an intracranial xenograft mouse using The Cancer Genome Atlas revealed that both MNK genes model, improved overall survival was observed in merestinib- (MKNK1 and MKNK2) are upregulated in mesenchymal glioblas- treated mice. Taken together, these data provide strong preclinical toma multiforme as compared with other subtypes. Expression of evidence that pharmacologic MNK inhibition targets mesenchy- MKNK1 is associated with increased glioma grade and correlated mal glioblastoma multiforme and its GSC population. with the mesenchymal GSC marker, CD44, and coexpression of MKNK1 and CD44 predicts poor survival in glioblastoma multi- Implications: These ﬁndings raise the possibility of MNK forme. In established and patient-derived cell lines, pharmaco- inhibition as a viable therapeutic approach to target the mes- logic MNK inhibition using LY2801653 (merestinib) inhibited enchymal subtype of glioblastoma multiforme. Mol Cancer Res; phosphorylation of the eukaryotic translation initiation factor 4E, 14(10); 984–93. 2016 AACR.

Introduction forme (2). A subpopulation of cancer stem cells, referred to as tumor-initiating cells (TIC) or glioma stem cells (GSC), has been Glioblastoma is the most common and deadliest primary brain identified in glioblastoma multiforme and other high-grade gli- tumor (1). Despite surgical resection, chemotherapy and radia- omas (3–6). GSCs expressing a mesenchymal gene signature are tion, there are no effective treatments for glioblastoma multi- particularly resistant to therapy, grow more rapidly than other subtypes, and express specific cancer stem cell markers (e.g., CD44; refs. 7–9). Developing strategies to target this resistant 1 Robert H. Lurie Comprehensive Cancer Center, Feinberg School of subpopulation of cells may lead to improved clinical outcomes. Medicine, Northwestern University, Chicago, Illinois. 2Department of Neurological Surgery, Feinberg School of Medicine, Northwestern Protein synthesis is a highly regulated process that contributes University,Chicago, Illinois. 3Department of Preventive Medicine, Fein- to oncogenesis and therapeutic resistance in glioblastoma multi- berg School of Medicine, Northwestern University, Chicago, Illinois. forme and other cancers (10–12). MNKs regulate protein synthe- 4Division of Hematology/Oncology, Department of Medicine, Fein- berg School of Medicine, Northwestern University, Chicago, Illinois. sis through phosphorylation of the eukaryotic translation initia- 5Division of Hematology/Oncology/Stem Cell Transplantation, tion factor 4E (eIF4E), a member of the eIF4F cap-binding Department of Pediatrics, Ann & Robert H. Lurie Children's Hospital complex (13, 14). Phosphorylation of eIF4E by MNKs leads to of Chicago, Chicago, Illinois. 6Department of Neurosurgery and Com- prehensive Cancer Center, University of Alabama at Birmingham, translation of a subset of oncogenic transcripts (15). Inhibition of Birmingham, Alabama. 7Department of Pathology, Feinberg School MNKs with small-molecule inhibitors or knockdown of MKNK1 of Medicine, Northwestern University, Chicago, Illinois. 8Department and MKNK2 disrupts growth of glioblastoma multiforme cells of Medicine, Jesse Brown VA Medical Center, Chicago, Illinois. and prevents tumor growth in vivo (16, 17). However, few clin- Note: Supplementary data for this article are available at Molecular Cancer ically relevant MNK inhibitors are available and none have been Research Online (http://mcr.aacrjournals.org/). shown to disrupt the growth of glioblastoma multiforme tumors Corresponding Author: Leonidas C. Platanias, The Robert H. Lurie Comprehen- in intracranial mouse models of the disease (10). sive Cancer Center of Northwestern University, 303 East Superior Street, Lurie- Merestinib (LY2801653) is a novel multikinase inhibitor, with 3125, Chicago, IL 60611-3008. Phone: 312-503-4267; Fax: 312-908-1372; Email: potent in vitro activity against MNKs, MET, and other protein [email protected] kinases (18–21). The compound has shown significant antitumor doi: 10.1158/1541-7786.MCR-16-0172 activity in several xenograft mouse models of non–small cell lung 2016 American Association for Cancer Research. cancer and other solid tumors, including one subcutaneous

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xenograft model of glioblastoma multiforme (20). In this study, developed with WesternBright ECL HRP substrate (Advansta) we sought to investigate MNKs as potential targets in GSCs. Our and autoradiography film (Denville Scientific). study suggests an important role for the MNK inhibitor, merestinib, as it inhibits MNK signaling in glioblastoma multiforme Polysomal fractionation and RT-PCR cells and GSCs, blocks growth of GSCs as neurospheres, and For polysomal fractionation, cell lysates were separated with a improves overall survival in an intracranial xenograft mouse 10% to 50% sucrose gradient as described previously (23). Poly- fi fi model. These ndings suggest a mesenchymal-speci c role for somal fractions were pooled and RNA was purified using the MNKs in glioblastoma multiforme and highlight a particular AllPrep RNA/Protein Kit (Qiagen). Specific primers for CCND1, vulnerability of mesenchymal GSCs for pharmacologic MNK CCND2, BCL2, and GAPDH (Thermo Fisher) were used for qRT- inhibition. PCR. GAPDH was used for normalization. Our results show that merestinib blocks phosphorylation of eIF4E in established glioblastoma multiforme cell lines and patient-derived GSCs. Analysis of data from The Cancer Genome Preprocessing of TCGA glioblastoma multiforme exon-array Atlas (TCGA) reveals that the MKNK1 and MKNK2 genes are data and subtyping overexpressed in glioblastoma multiforme from the mesenchy- The unprocessed Affymetrix exon-array datasets for 419 glio- mal subtype. Furthermore, in glioblastoma multiforme, MKNK1 blastoma multiforme samples were downloaded from the TCGA expression correlates with CD44, a mesenchymal GSC marker. data portal (https://tcga-data.nci.nih.gov/tcga). We followed the Using patient-derived mesenchymal GSCs, we found that mer- data preprocessing procedure described in our recent study (24). estinib disrupts cancer stem cell viability and frequency, as deter- Samples underwent subtyping into one of four molecular classes mined by neurosphere formation and extreme limiting dilution of glioblastoma multiforme (classical, mesenchymal, proneural, analysis (ELDA). Finally, in an intracranial xenograft mouse or neural), as described previously (25). We used an isoform- model of glioblastoma multiforme, merestinib inhibited MNK based classifier to obtain the patient subtype information (24). signaling and improved overall survival. Unpaired t tests were used to determine whether MKNK1, MKNK2,orMET were differentially expressed between different glioblastoma multiforme subtypes. Materials and Methods Cell culture and reagents Analysis of TCGA glioblastoma multiforme and LGG RNA-seq Glioblastoma multiforme cell lines were grown in DMEM data supplemented with FBS (10%) and gentamycin (0.1 mg/mL). RNASeqV2 level 3–released gene level expression data for RNA U87 cells were authenticated by short tandem repeat (STR) sequencing (RNA-seq) were downloaded for glioblastoma multi- analysis in January 2016 (Genetica DNA Laboratories). The forme and low-grade gliomas (LGG) from TCGA. The data pro- isolation of patient-derived glioma stem cells and generation cessing and quality control were done by the Broad Institute's of GSC lines (83Mes, MD30, and GBM43) has been described TCGA workgroup. The reference gene transcript set was based on previously (8, 22). GSCs were cultured in DMEM/F12 supple- the HG19 UCSC gene standard track. MapSplice (26) was used to mented with EGF (20 ng/mL), bFGF (20 ng/mL), heparin (5 perform the alignment and RSEM (27) to perform the quantita- mg/mL), B27 (2%), and gentamycin (0.1 mg/mL). Merestinib tion. Unpaired t tests were used to determine whether MKNK1 was was provided by Eli Lilly & Company and dissolved in DMSO differentially expressed between LGG and glioblastoma multi- for in vitro studies. For in vivo studies, merestinib was first forme. The upper quartile normalized RSEM count estimates were dissolved in PEG400, followed by sonication and addition of base-10 log transformed before t tests. 20% Captisol in water.

Immunoblotting and antibodies Analysis of TCGA expression data and multigene prognostic Cells were harvested and washed three times with cold PBS by index MKNK1 centrifugation. Cell pellets were lysed with phosphorylation lysis gene expression data were downloaded from the GBM Bio Discovery Portal (GBM-BioDP) as previously described using buffer (50 mmol/L Hepes, 150 mmol/L NaCl, 1 mmol/L MgCl2, 0.5% Triton, 10% glycerol, 0.5% sodium deoxycholate, pH 7.9) the Verhaak Core dataset (25, 28). Survival analysis of TCGA supplemented freshly with phosphatase and protease inhibitors patients was performed using the multigene prognostic index (Roche). Protein concentrations were measured by Bradford assay from the GBM-BioDP. For survival analysis, gene expression data MKNK1 CD44 (Bio-Rad) using the Synergy HT plate reader and Gen5 software for and (Agilent G4502A_07) for patients from the (BioTek Instruments). Equal concentrations of whole-cell Verhaak Core were used. lysates were separated by SDS-PAGE (Bio-Rad) and transferred by semidry transfer to Immobilon PVDF membranes (Milli- Cell viability assay pore). Membranes were blocked with 5% BSA in 1 TBST and To determine cell viability following treatment with meresti- incubated with primary antibodies overnight. Primary antibo- nib, the Cell Proliferation Reagent WST-1 (Roche) was used dies against phospho-eIF4E (Ser209) and eIF4E were obtained according to the manufacturer's instructions. Brieﬂy, U87, 83Mes, from Cell Signaling Technology and used at a dilution of MD30, or GBM43 cells were seeded into 96-well plates at a density 1:1,000. Following primary antibody incubation, membranes of 3,000 cells per well in the presence of DMSO or merestinib at were washed three times with 1 TBST and incubated with anti- the indicated concentrations. After 5 days of incubation at 37Cin rabbit (GE Healthcare) or anti-mouse (Bio-Rad) horseradish 5% CO2, the WST-1 reagent was added and viability was quan- peroxidase (HRP)-conjugated secondary antibodies for 1 hour. tiﬁed using a Synergy HT plate reader and the Gen5 software Membranes were then washed three times with 1 TBST and (BioTek).

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Soft agar assay processed using the BOND-MAX Automated IHC/ISH Stainer To assess colony formation, the CytoSelect 96-Well Cell Trans- and its Polymer Detection System (Leica Biosystems). The Bond formation Assay (Cell Biolabs) was used according to the man- Dewax Solution (AR9222) was used at 72C. The Bond Epitope ufacturer's instructions. Briefly, U87, 83Mes, MD30, or GBM43 Retrieval Solution 1 (AR9961) was used for 20 minutes at cells were seeded in soft agar at a density of 2,500 cells per well in 100C. Samples were pretreated with 3% hydrogen peroxide the presence of DMSO or merestinib (10 mmol/L). After 7 days of for 5 minutes. For detection of eIF4E phosphorylation, the incubation at 37 Cin5%CO2, colony formation was quantified eIF4E (Ser209) antibody [EP2151Y] (Abcam) was used at a by solubilizing soft agar, lysing cells, and incubating cell lysates 1:2,000 dilution for 15 minutes. The post primary polymer with the CyQUANT GR Dye (Cell Biolabs), followed by analysis penetration enhancer reagent was added for 8 minutes, fol- with the Synergy HT plate reader and the Gen5 software (BioTek). lowed by the polymer poly-HRP secondary antibody for 8 minutes. Hematoxylin was used for 5 minutes. eIF4E phos- fi Apoptosis assay phorylation (Ser209) was semiquanti ed by light microscopic fi For analysis of apoptosis, the BD Pharmingen FITC Annexin V analysis, in which a board-certi ed neuropathologist (C. Hor- Apoptosis Detection Kit I (BD Biosciences) was used according to binski) ranked the tumors from strongest to weakest, followed the manufacturer's instructions. Briefly, U87 cells were seeded by Spearman rank order correlation. Mitoses were scored per 10 high-power fields (600). Both phospho-eIF4E and mitotic into 6-well plates and incubated at 37 Cin5%CO2 until reaching 70% confluence. Cells were then incubated with DMSO or mer- index were analyzed while blinded to treatment group. estinib (10 mmol/L) for 48 hours. Following treatment, cells were harvested using trypsin, washed three times with PBS, and stained Statistical analysis with propidium iodide (PI) staining solution and FITC Annexin V. Unless otherwise specified, statistics were performed using Stained samples were analyzed by flow cytometry and FlowJo 10 GraphPad Prism 6.0 for Mac. for Mac. Results Neurosphere assay and ELDA Recent advances in cancer genomics have identified at least 4 Cells were seeded into round-bottom 96-well plates (Greiner distinct molecular subtypes of glioblastoma multiforme: classical, Bio-One) containing DMSO or merestinib at the indicated cell mesenchymal, neural, and proneural (25, 32, 33). These subtypes numbers by flow cytometry using forward- and side scatter, single- were initially categorized using gene expression profiling, which cell sorting as described previously (8). After 7 days, cells were identified expression signatures similar to those found during stained with 0.1 mg/mL acridine orange as described previously normal neurogenesis. Our recent studies have revised subtype (29). Neurospheres were imaged using the Cytation 3 Cell Imag- classification using isoform-level gene expression values, which ing Multi-Mode Reader with a 4 objective. For ELDA, neuro- provide more robust subtype classifications with greater prognos- sphere diameters were measured using the Cytation 3 software. tic significance (24). We sought to use our subtype classifications Neurospheres measuring 100 mm in diameter were scored to understand how the MNK genes (MKNK1 and MKNK2) are positively for sphere formation for ELDA and analyzed using the differentially expressed in glioblastoma multiforme. Using RNA- ELDA online software (http://bioinf.wehi.edu.au/software/elda/; seq data from TCGA, we discovered that MKNK1 and MKNK2 are ref. 30). significantly overexpressed in mesenchymal subtype glioblastoma multiforme when compared with other subtypes (Fig. 1A and Animal studies B). MET, a previously identified mesenchymal gene and target of All animal studies were carried out in accordance with the merestinib, was also enriched in the mesenchymal cohort (Fig. Institutional Animal Care and Use Committee of the Northwest- 1C; refs. 19, 34). ern University (Chicago, IL). Luciferase-expressing U87 cells were Given the importance of MNK1 in the maintenance of glio- m intracranially injected (100,000 cells/ L with a total injection blastoma multiforme survival under various conditions (11, 17), m volume of 2 L/animal) into 5- to 6-week-old athymic nu/nu we further explored the relationship between MNK1, molecular female mice (Taconic Biosciences). Bioluminescence imaging was subtype, and glioma grade. Using data from three different gene used to monitor tumor growth as described previously (31). At 17 arrays, we validated that MKNK1 is overexpressed in the mesen- days postinjection of tumor cells, mice were randomized into chymal subtype (Fig. 1D–F). We also found that MKNK1 expres- control and treatment groups according to intracranial tumor sion increases with glioma grade and is highest in glioblastoma bioluminescence values. Mice were treated with vehicle control or multiforme, when compared with grade 2 or grade 3 gliomas (Fig. merestinib at a dose of 12 mg/kg, twice daily (5 days of treatment 1G). These data align with findings by others indicating that and 2 days of rest) for 2 weeks. Mice were monitored until MKNK1 expression is increased in glioblastoma multiforme when required euthanasia due to indication of neurologic compromise compared with normal human astrocytes or patients with oligo- from increasing tumor burden or at 55 days after tumor cell dendroglioma or anaplastic astrocytoma (17). Furthermore, injection. across all four glioblastoma multiforme subtypes, MKNK1 and the mesenchymal GSC marker, CD44, are positively correlated H&E staining and IHC and predict poor prognosis when overexpressed concurrently (Fig. After sacrificing mice, resected brains were harvested for hema- 1H and I; ref. 8). Taken together, these findings indicate an toxylin and eosin (H&E) staining and immunohistochemical important role for MNK1 signaling in the mesenchymal subtype analysis. Brains were fixed with 10% buffered formalin overnight. of glioblastoma multiforme and maintenance of mesenchymal Brains were then embedded in paraffin and sectioned for H&E GSCs, suggesting MNKs are promising targets in this glioblastoma staining and immunohistochemical analysis. Sections were multiforme subtype.

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A BC **** **** **** **** **** **** **** **** **** 5.5 6.5 8 7 5.0 6.0 6 4.5 5.5 5 4.0 5.0 4 3 4.5 3.5 2

4.0 MET log2 Expression 1

3.0 MKNK2 log2 Expression MKNK1 log2 Expression CL M N PN CL M N PN CL M N PN

D EF AgilentG4502A_07 HuEx-1_0-st-v2 HT_HG-U133A **** **** **** 2 **** ** 1.5 **** * 2.0 ****

1 1.0 1.5 1.0 0 0.5 0.5 Expression z-score Expression z-score − Expression z-score 1 0.0 0.0

−2 −0.5 −0.5 CL M N PN CL M N PN CL M N PN MKNK1 MKNK1 MKNK1

G **** HIMultigene prognostic index **** (MKNK1 and CD44) 3.2 9 C 100 Prog. Index 8 M Below med 3.0 N 80 Above med P 2.8 7 60 Log-rank P = 0.026 2.6 6 2.4 5 40 log2 Expression

4 Survival (%) 2.2 20 r 2.0 3 Pearson = 0.4020;**** 2 Spearman ρ = 0.3614;**** 0 1.8 CD44

MKNK1 log10 Expression 0 3.0 3.5 4.0 4.5 5.0 5.5 6.0 500 1,0001,5002,0002,5003,0003,500 GBM MKNK1 log2 expression Grade 2 Grade 3 Survival in days

Figure 1. Enhanced MNK mRNA expression in mesenchymal subtype GBM. A–C, MKNK1, MKNK2, and MET RNA-seq data were analyzed using a TCGA cohort of patients 40 years old with classical (CL), mesenchymal (M), neural (N), and proneural (PN) subtype glioblastoma multiforme (GBM). Unpaired two-tailed t tests: , P 0.0001. D–F, MKNK1 expression z-score data from TCGA were downloaded from the GBM-BioDP for CL, M, N, and PN subtype glioblastoma multiforme from the Agilent (AgilentG4502A_07), Human Exon (HuEx-1_0-st-v2), and HT Human Genome U133 (HT_HG-U133A) arrays (http://gbm-biodp.nci.nih.gov). Unpaired two-tailed t tests: , P 0.05; , P 0.01; , P 0.0001. G, MKNK1 RNA-seq data were analyzed using a TCGA cohort of patients 40 years old with grade 2 gliomas, grade 3 gliomas, and glioblastoma multiforme. Unpaired two-tailed t test: , P 0.0001. H, MKNK1 and CD44 RNA-seq data from a TCGA cohort of patients 40 years old were analyzed by linear regression analysis: , P 0.0001. I, MKNK1 and CD44 Agilent gene expression data from TCGA were used for multigene prognostic index. Figure was generated using the GBM-BioDP software.

To study the effectiveness of MNK inhibition in glioblastoma refs. 7–9, 22). The patient-derived cell lines were grown as non- multiforme, we employed one established cell line (U87) and adherent neurospheres in serum-free medium and were desig- three patient-derived GSC cell lines (83Mes, GBM43, and MD30; nated "glioma stem cells" (GSC). GSCs have unique properties,

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including the ability to form neurospheres, and are enriched for the effects of merestinib on this process. To this end, we the mesenchymal GSC markers aldehyde dehydrogenase and analyzed monosomal and polysomal fractions. Treatment of CD44 (Supplementary Fig. S1). We have extensively characterized U87, 83Mes, and GBM43 cells with the inhibitor resulted in an these GSCs in previous publications. altered translational profile, as demonstrated by an increase in Given the potential role for MNKs in glioblastoma multi- the monosomal (40S, 60S, and 80S) peaks and a decrease in the forme, we sought to study the effect of merestinib on MNK- polysomal peaks (Fig. 2D–F). Further analysis of U87 and mediated protein phosphorylation and mRNA translation in 83Mes profiles shows a decrease in the area under the poly- the established glioblastoma multiforme cell line, U87, and in somal curve upon treatment, indicating a decrease in global GSCs. The phosphorylation of eIF4E on serine 209 was blocked protein synthesis (Fig. 2G and H). When transcripts for cyclins by merestinib in U87 as well as the patient-derived GSC lines D1 and D2 undergoing active translation were analyzed, we 83Mes and GBM43 (Fig. 2A–C). As eIF4E phosphorylation is found that mRNA levels for both these cyclins were significantly important for active mRNA translation, we sought to determine decreased in the polysomal fractions of merestinib-treated

ABC U87 83Mes GBM43 Mer (mmol/L): 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 eIF4E eIF4E eIF4E Blot: anti-pSer209 eIF4E Blot: anti-pSer209 eIF4E Blot: anti-pSer209 eIF4E Figure 2. eIF4E eIF4E eIF4E Merestinib blocks MNK signaling Blot: anti-eIF4E Blot: anti-eIF4E Blot: anti-eIF4E and inhibits translation. A–C, U87 (A), 83Mes (B), and GBM43 (C) DEFcells were treated with increasing DMSO DMSO DMSO concentrations of merestinib Merestinib Merestinib I Merestinib I (MER) for 1 hour, as indicated. 0.7 1.0 Merestinib II 1.0 Merestinib II Equal amounts of whole-cell 0.6 80S lysates were resolved by SDS- 80S 0.8 0.8 PAGE and transferred to PVDF 80S 60S 0.5 40S membranes. Blots were probed 60S 0.6 60S 0.6 with an antibody against phospho- 0.4 40S 40S eIF4E (Ser209) followed by 0.3 0.4 0.4 Polysomes stripping and reprobing with an Polysomes O.D. 254 nm 254 O.D. 0.2 Polysomes antibody against eIF4E. D–F, U87 0.2 0.2 cells (D) were treated with DMSO 0.1 or merestinib (1 mmol/L) for 24 0.0 0.0 0.0 hours. 83Mes (E) and GBM43 (F) cells were treated with DMSO or Gradient depth Gradient depth Gradient depth merestinib at ﬁnal concentrations GHU87 83Mes of 1 mmol/L (I) or 10 mmol/L (II) for 24 hours. Cells were then subjected to hypotonic lysis and separated ** by a 10% to 50% sucrose gradient 100 * * 100 and the optical density (O.D.) at 75 75 254 nm was measured. The O.D. is displayed as a function of the 50 50 gradient depth. G and H, for U87 (G) and 83Mes (H) cells, the AUCs 25

(% control) 25 of polysomal and monosomal (% control) fractions were calculated using 0 0 ImageJ software. Relative polysomal/monosomal areas were Polysomal/monosomal area Mer area Polysomal/monosomal Mer I DMSO DMSO Mer II calculated for DMSO and IJDMSO merestinib-treated samples. I and DMSO J, for U87 (I) and 83Mes (J) cells, Merestinib Merestinib total mRNA from polysomal 100 * * 100 ** fractions was pooled, and fold change was determined by RT- 75 75 PCR using GAPDH for normalization. Data represent 50 50 means SEM of two independent experiments. Unpaired two-tailed t (% control) P P (% control) 25 25 test: , 0.05; , 0.01. Polysomal mRNA Polysomal Polysomal mRNA 0 0

BCL2 BCL2 CCND1 CCND2 CCND1 CCND2

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AB * 100 100

75 75

50 50

Figure 3. 25 25 Effects of merestinib on viability, colony formation, and apoptosis in glioblastoma multiforme. A, U87 cells were seeded into 96-well plates at a density of 3,000 cells per well with (% control) viability Cell 0 0 increasing concentrations of merestinib. After 5 days, cell Soft agar assay (% control) viability was quantified using the WST-1 assay. Data represent 0 0.3 1 3 10 means SEM of five independent experiments. B, U87 cells Merestinib (mmol/L) were seeded into 96-well plates at a density of 2,500 cells per DMSO well in soft agar with the indicated treatments. After 7 days, Merestinib colony formation was quantified using the fluorescent C 5 Q1 Q2 CyQUANT GR Dye. Data represent means SEM of three 10 7.28 3.27 independent experiments. Unpaired two-tailed t test: , P 4 15 0.05. C, U87 cells were seeded into 6-well plates containing 10 *

DMSO or merestinib, as indicated. After 2 days, cells were 3

PI 10 stained with propidium iodide (PI) and Annexin V-FITC. Representative dot plots from cells treated with DMSO or DMSO 102 Q4 Q3 10 merestinib are shown. Dot plots were generated using FlowJo 88.7 0.75 1 10 10. Data represent means SEM of three independent 102 103 104 105 experiments. Unpaired two-tailed t test: Annexin V-FITC , P 0.05. 5 Q1 Q2 5 10 5.83 3.40

104 Apoptotic cells (%) 103 PI 0 102 Q4 Q3 Merestinib 79.4 11.4 101 102 103 104 105 DMSO Annexin V-FITC Merestinib

samples (Fig. 2I and J), suggesting that merestinib is a potent neurosphere size across most cell densities (Fig. 4C–F). Further- inhibitor of the translation of these oncogenic mRNAs. Taken more, ELDA of neurospheres was used to determine effects on together, these findings indicate that merestinib treatment stem cell frequencies. Merestinib led to a significant decrease in inhibits MNK activity and protein synthesis in established the GSC frequency in 83Mes and MD30 GSCs. Specifically, the glioblastoma multiforme cells and GSCs. stem cell frequencies in 83Mes GSCs dropped from 1 in 3.45 for We next sought to identify the effects of merestinib on glio- DMSO to 1 in 30.24 cells for merestinib (Fig. 4G). Similarly, the blastoma multiforme cells. For these studies, the established stem cell frequencies for MD30 GSCs dropped from 1 in 16.6 for glioblastoma multiforme cell line, U87, was used. Cell viability, DMSO to 1 in 288.7 cells for merestinib (Fig. 4H). These results anchorage-independent growth in soft agar, and apoptosis were indicate a significant decrease in the cancer stem cell populations. assessed following treatment with merestinib (Fig. 3). The inhib- Taken together, these results strongly suggest that merestinib itor exhibited suppressive effects on cell viability and anchorage- disrupts these tumor-initiating cells in mesenchymal subtype independent growth in U87 cells (Fig. 3A and B). In addition, gliomas. there was an increase in apoptosis following 48-hour incubation In further studies, we tested the inhibitor in an intracranial with the inhibitor (Fig. 3C). We then measured the effect of xenograft glioblastoma multiforme mouse model. Nude mice merestinib on cell viability, anchorage-independent growth, and were injected with luciferase-expressing U87 cells, and after neurosphere formation on GSCs. Merestinib treatment decreased tumor formation, mice were treated with either merestinib or cell viability in a dose-dependent manner in 83Mes, MD30, and control vehicle for two weeks and monitored for a total of 55 GBM43 GSCs, with IC50 values of 4.3, 4.9, and 3.2 mmol/L, days. Comparison of control and merestinib treated mice respectively (Fig. 4A). Similarly, merestinib disrupted malignant shows a trend toward decreased tumor volume (Fig. 5A and transformation as measured by anchorage-independent growth in B). Furthermore, merestinib treatment significantly prolonged soft agar (Fig. 4B). We next examined whether merestinib could survival when compared with the vehicle control (Fig. 5C). disrupt neurosphere formation in 83Mes and MD30 GSCs. When Analysis of tumors by IHC demonstrated a decrease in eIF4E increasing numbers of cells were seeded, merestinib disrupted phosphorylation, indicating that merestinib was able to inhibit

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A B

) μ 83Mes 83Mes; IC50 = 4.3 mol/L ol

tr μ *** * * MD30 100 MD30; IC50 = 4.9 mol/L 100 on GBM43; IC = 3.2 μmol/L GBM43 75 50 75

50 50 ty (% c

bili 25 25 via

ll 0 0

Ce 013 10 − + − + − +

m assay (% control) agar Soft Merestinib ( mol/L) Merestinib 83Mes MD30 C D

DMSO DMSO

Merestinib Merestinib 1 5 10 50 100 1 5 10 50 100 EFCells seeded per well Cells seeded per well 100 DMSO ** 100 DMSO **** Merestinib Merestinib 75 75 ** 50 50 ** (% control) (% control) 25 25 * * NS cross-sectional area NS cross-sectional area 0 0 1 5 10 50 100 1 5 10 50 100 Cells seeded per well Cells seeded per well G H 0.0 DMSO 0.0 Merestinib **** −0.5 −0.5

−1.0 −1.0

− 1.5 −1.5 DMSO Merestinib −2.0 −2.0 Log fraction without spheres Log fraction without spheres **** −2.5 −2.5 020406080100 0 20406080100 Cells seeded per well Cells seeded per well 1/(stem cell frequency) 1/(stem cell frequency) DMSO: 1/3.45 DMSO: 1/16.6 Merestinib: 1/30.24 Merestinib: 1/288.7

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A Vehicle Merestinib D Vehicle Merestinib 6.00 × 109

Day 0 H&E

0.35 × 108 × 9 Figure 5. 9.00 10 Merestinib blocks MNK signaling and improves survival in an intracranial Day 3 glioblastoma multiforme xenograft. A and B, nude mice were injected with luciferase- expressing U87 cells by intracranial 0.70 × 108 p-eIF4E injection. On days 0 and 3 of treatment, bioluminescence imaging (BLI) was B E performed in vehicle and merestinib- p-eIF4E IHC treated mice. BLI pictures from one vehicle- 8 P = 0.14 Rank order treated and one merestinib mouse with (1 = strongest, Treatment similar starting values are shown. Graph 6 8 = weakest) group represents means SEM of day 3 BLI values 1 Vehicle normalized to day 1 values. Unpaired two- 2 Vehicle tailed t test: P ¼ 0.14. C, survival analysis of 4 vehicle (n ¼ 10) and merestinib (n ¼ 11) 3 Vehicle treated mice. Log-rank (Mantel–Cox) test: 4 Vehicle P ¼ 0.0295. Red arrows, 2 treatment cycles 2 5 Merestinib (5 days of treatment, 2 days of rest). D, Normalized BLI 6 Vehicle immunohistochemical staining of brain 0 7 Merestinib tumors from vehicle and merestinib-treated 8 Merestinib mice are shown. H&E staining (top) and IHC Vehicle Merestinib Spearman correlation, P = 0.046 for phopsho-eIF4E (Ser209; bottom) are shown. Scale bar, 50 mm. E, rank order of IHC C F for phospho-eIF4E (Ser209) in vehicle and 12 P merestinib treated samples is shown. 100 Vehicle = 0.06 P ¼ Spearman rank correlation (two-tailed): Merestinib 0.046. F, number of mitoses per 10 high- 80 ﬁ 8 power elds (hpf) in brain tumors from P = 0.0295 vehicle and merestinib-treated mice are 60 shown. Unpaired two-tailed t test: P ¼ 0.06. 40 4

Survival (%) 20 Mitoses per 10 hpf 0 0 0 20 25 30 35 40 45 50 55 Vehicle Merestinib Days postinjection

MNK signaling in vivo (Fig. 5D). Rank order analysis of mer- Discussion estinib-treated or control samples demonstrated a signiﬁcant Most glioblastoma multiforme patients die from tumor recur- decrease in eIF4E phosphorylation in the treated group (Fig. rence after standard therapy. Therefore, better treatment options 5E). Finally, the number of mitoses per 10 high-power ﬁelds for malignant brain tumors are desperately needed. New therapies showed a trend toward reduced proliferation in treated samples for glioblastoma multiforme and other high-grade gliomas must when compared with controls (Fig. 5F). include targeting of resistant GSC populations to prevent tumor

Figure 4. Merestinib inhibits GSCs. A, 83Mes, MD30, and GBM43 cells were seeded into 96-well plates at a density of 3,000 cells per well with increasing concentrations of merestinib. After 5 days, cell viability was quantified using the WST-1 assay. Data represent means SEM of three independent experiments. B, 83Mes, MD30, and GBM43 cells were seeded into 96-well plates at a density of 2,500 cells per well in soft agar containing DMSO or merestinib. After 7 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Data represent means SEM of three independent experiments. Unpaired two-tailed t test: , P 0.05; , P 0.001. C and D, 83Mes and MD30 cells were seeded in duplicate into round-bottom 96-well plates containing merestinib (10 mmol/L) by forward- and side scatter, single-cell sorting at the indicated cell densities. After 7 days, neurospheres were stained with acridine orange and imaged using a Cytation 3 Cell Imaging Multi-Mode Reader with a 4 objective. Scale bar, 1,000 mm. Representative images for DMSO and merestinib treatment are shown. E–F, cross-sectional areas of 83Mes (E) and MD30 (F) neurospheres (NS) were measured using the Cytation 3 software. Data represent means SEM of three independent experiments. Unpaired two-tailed t test: , P 0.05; , P 0.01; , P 0.0001. G and H, ELDA for 83Mes (G) and MD30 (H) neurospheres was performed using the ELDA software (http://bioinf.wehi.edu.au/software/elda/) with 6 technical replicates. Statistics for stemcell frequencies of DMSO and merestinib treated samples are shown. c2: , P 0.0001.

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Bell et al.

recurrence and improve clinical outcomes. Furthermore, treat- merestinib at low doses (20). Therefore, the diversity of mer- ment strategies should be tailored for particular molecular estinib targets could prove beneficial, preventing the develop- subtypes. Mesenchymal subtype glioblastoma multiforme are ment of resistance by redundant or parallel signaling pathways among the most aggressive forms of the tumor with a median (36–38). survival as low as 11.8 months (25). Mesenchymal tumors and Our findings provide strong evidence for targeting the MNK GSCs are enriched with particular molecular markers (e.g., axis in glioblastoma multiforme tumors. However, the specific MET, CD44) and exhibit increased proliferation rates, increased mechanism by which MNKs support the growth of GSCs radiation resistance, and poorer overall survival when com- remains elusive. Some studies have suggested that MNK1 pared with other subtypes (35). Developing therapies to target positively regulates expression of the GSC-promoting factors mesenchymal GSCs is an attractive approach that has the TGFb and Sema3C (17). In mesenchymal GSCs, TGFb signaling potential to improve outcomes in glioblastoma multiforme has been shown to regulate proliferation, invasion, and pro- and other high-grade gliomas. mote immune evasion (17, 39). Furthermore, GSC secretion of In this study, we present several novel findings.First,weuse the soluble factor, Sema3C, promotes cancer stem cell main- our previously described isoform-level, gene expression classi- tenance through activation of Rac1 signaling (40). Similarly, fication system (24) to provide evidence that the MNK genes MNK signaling plays a key role in the maintenance of stem cell (MKNK1 and MKNK2) are differentially expressed across glio- populations in other cancer types. In leukemia, the MNK–eIF4E blastoma multiforme subtypes and are most highly expressed signaling cascade has been implicated in the maintenance of in mesenchymal glioblastoma multiforme. Expanding upon leukemic precursors in blast crisis chronic myeloid leukemia this finding, we demonstrated that MKNK1 expression is (41). In line with these findings, we have shown that MNK increased in glioblastoma multiforme as compared with grade inhibition with cercosporamide, an antifungal compound 2 and grade 3 gliomas. MKNK1 expression also correlated with found to be a potent inhibitor of MNK1 and MNK2 activity the previously identified mesenchymal GSC gene, CD44,and (42), disrupts colony formation in primary leukemic progeni- simultaneous upregulation of both genes predicts poor prog- tors from patients with acute myeloid leukemia (43). Taken nosis in glioblastoma multiforme. These findings led us to together, these observations strongly suggest a role for MNKs in explore the potential of a multikinase inhibitor, merestinib, the maintenance of therapy-resistant cancer stem cell popula- whichhasshownpotentin vitro activity against MNKs (20), as a tions. Further studies are warranted and may show important therapy to eliminate mesenchymal GSCs. Our findings dem- clinical–translational implications for the treatment of glio- onstrate that merestinib blocks MNK signaling in an estab- blastoma multiforme. lished glioblastoma multiforme cell line and three GSC lines. Furthermore, merestinib significantly reduced global protein Disclosure of Potential Conflicts of Interest synthesis and inhibited translation of the oncogenic mRNAs, No potential conflicts of interest were disclosed. CCND1 and CCND2, and produced a small decrease in BCL2 mRNA in glioblastoma multiforme cells and GSCs. The inhibitor demonstrated activity in viability, soft agar, and apoptosis Authors' Contributions assays using the established glioblastoma multiforme cell line, Conception and design: J.B. Bell, F.D. Eckerdt, L.C. Platanias U87. Furthermore, merestinib showed potent activity against Development of methodology: J.B. Bell, F.D. Eckerdt, Y. Bi, A.A. Alvarez, GSC viability, growth in soft agar, and neurosphere growth, C. Horbinski, C.D. James, L.C. Platanias indicating an inhibition on the growth potential of these cells. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.B. Bell, F.D. Eckerdt, K. Alley, L.P. Magnusson, Using ELDA, we also demonstrated that merestinib disrupts the H. Hussain, Y. Bi, A.D. Arslan, J. Clymer, S.-Y. Cheng, I. Nakano, C. Horbinski sphere-forming potential of two mesenchymal GSC lines, indi- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cating a suppression of the stem cell population. Finally, in an computational analysis): J.B. Bell, F.D. Eckerdt, K. Alley, Y. Bi, A.A. Alvarez, intracranial xenograft mouse model of glioblastoma multi- C. Horbinski, R.V. Davuluri, C.D. James, L.C. Platanias forme, we found that merestinib disrupts MNK signaling in Writing, review, and/or revision of the manuscript: J.B. Bell, F.D. Eckerdt, vivo and significantly prolongs survival in mice. K. Alley, H. Hussain, A.A. Alvarez, S. Goldman, S.-Y. Cheng, C. Horbinski, R.V. Davuluri, L.C. Platanias Merestinib is a multikinase inhibitor with activity against Administrative, technical, or material support (i.e., reporting or organizing both MNK1 and MNK2 and has demonstrated potent antineo- data, constructing databases): L.P. Magnusson, H. Hussain, C. Horbinski plastic effects in several solid tumors (18–20). For the first time, Study supervision: L.P. Magnusson, C.D. James, L.C. Platanias our study provides detailed analysis of this inhibitor in glioblastoma multiforme and establishes an important effect on Grant Support cancer stem cells. The striking effect of the inhibitor on GSC This work was supported by the NIH grants CA155566, CA77816, and growth, malignant transformation and neurosphere formation CA121192, and by grant I01CX000916 from the Department of Veterans is particularly interesting, as it raises the possibility of a prom- Affairs. J.B. Bell was supported in part by NIH/NCI training grant T32 ising approach for targeting the tumor-initiating cancer stem CA09560 and MSTP NIH training grant T32 GM008152. A.D. Arslan cell population. Although MNK1 and MNK2 are the only and A.A. Alvarez were supported in part by NIH/NCI training grant T32 serine/threonine kinases inhibited by merestinib, it is impor- CA070085. tant to note that the inhibitor affects other kinases (20). In vitro The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked assays have shown that the inhibitor blocks the activity of other advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate protein kinases known to be important for the growth of this fact. glioblastoma multiforme and GSCs. In particular, the receptor tyrosine kinases MET and AXL, which have also been identified Received May 12, 2016; accepted June 11, 2016; published OnlineFirst as drivers of mesenchymal GSCs (8, 9), are inhibited by June 30, 2016.

992 Mol Cancer Res; 14(10) October 2016 Molecular Cancer Research

MNK Inhibition in Glioma Stem Cells

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MNK Inhibition Disrupts Mesenchymal Glioma Stem Cells and Prolongs Survival in a Mouse Model of Glioblastoma

Jonathan B. Bell, Frank D. Eckerdt, Kristen Alley, et al.

Mol Cancer Res 2016;14:984-993. Published OnlineFirst June 30, 2016.

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