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

Abstract

Glioblastoma multiforme remains the deadliest malignant brain tumor, with glioma stem cells (GSC) contributing to treatment resistance and tumor recurrence. We have identified MAPK-interacting kinases (MNK) as potential targets for the GSC population in glioblastoma multiforme. Isoform-level subtyping using The Cancer Genome Atlas revealed that both MNK genes (MKNK1 and MKNK2) are upregulated in mesenchymal glioblastoma multiforme as compared with other subtypes. Expression of MKNK1 is associated with increased glioma grade and correlated with the mesenchymal GSC marker, CD44, and coexpression of MKNK1 and CD44 predicts poor survival in glioblastoma multiforme. In established and patient-derived cell lines, pharmacologic MNK inhibition using LY2801653 (merestinib) inhibited phosphorylation of the eukaryotic translation initiation factor 4E, a crucial effector for MNK-induced mRNA translation in cancer cells and a marker of transformation. Importantly, merestinib inhibited growth of GSCs grown as neurospheres as determined by extreme limiting dilution analysis. When the effects of merestinib were assessed in vivo using an intracranial xenograft mouse model, improved overall survival was observed in merestinib-treated mice. Taken together, these data provide strong preclinical evidence that pharmacologic MNK inhibition targets mesenchymal glioblastoma multiforme and its GSC population.

Implications: These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of glioblastoma multiforme. Mol Cancer Res; 14(10); 984-93. ©2016 AACR.

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 GBM. Unpaired two-tailed t-tests, ****p≤0.0001. D–F, MKNK1 expression z-score data from TCGA was downloaded from the GBM-BioDP for CL, M, N, and PN subtype GBM 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 was analyzed using a TCGA-cohort of patients ≥40 years old with Grade 2 gliomas, Grade 3 gliomas, and GBM. 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 multi-gene prognostic index. Figure was generated using the GBM-BioDP software.

Figure 2. Merestinib blocks MNK signaling and inhibits translation

A–C, U87 (A), 83Mes (B), and GBM43 (C) cells were treated with increasing concentrations of merestinib for 1 hour, as indicated. Equal amounts of whole cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Blots were probed with an antibody against phospho-eIF4E (Ser209) followed by stripping and re-probing with an antibody against eIF4E. D–F, U87 cells (D) were treated with DMSO or merestinib (1 μM) for 24 hours. 83Mes (E) and GBM43 (F) cells were treated with DMSO or merestinib at final concentrations of 1μM (I) or 10 μM (II) for 24 hours. Cells were then subjected to hypotonic lysis and separated by a 10–50% sucrose gradient and the O.D. at 254nm was measured. The O.D. is displayed as a function of the gradient depth. G–H, For U87 (G) and 83Mes (H) cells, the areas under the curves of polysomal and monosomal fractions were calculated using ImageJ software. Relative polysomal/monosomal areas were calculated for DMSO and merestinib treated samples. I–J, For U87 (I) and 83Mes (J) cells, total mRNA from polysomal fractions was pooled and fold change was determined by RT-PCR using GAPDH for normalization. Data represent means + SEM of two independent experiments. Unpaired two-tailed t-test, *p≤0.05, **p≤0.01.

Figure 3. Effects of merestinib on viability, colony formation, and apoptosis in GBM

A, U87 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 five independent experiments. B, U87 cells were seeded into 96-well plates at a density of 2,500 cells per well in soft agar with the indicated treatments. 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. C, U87 cells were seeded into 6-well plates containing DMSO or merestinib, as indicated. After 2 days, cells were stained with Propidium Iodide (PI) and Annexin V-FITC. Representative dot plots from cells treated with DMSO or merestinib are shown. Dot plots were generated using FlowJo 10. Data represent means + SEM of three independent experiments. Unpaired two-tailed t-test, *p≤0.05.

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–D, 83Mes and MD30 cells were seeded in duplicate into round-bottom 96-well plates containing merestinib (10 μM) 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 4X objective. Scale bar = 1000 μm. Representative images for DMSO and merestinib treatment are shown. E–F, Cross-sectional areas of 83Mes (E) and MD30 (F) neurospheres were measured using the Cytation 3 software. Data represent means of ± SEM of three independent experiments. Unpaired two-tailed t-test, *p≤0.05, **p≤0.01, ****p≤0.0001. G–H, Extreme limiting dilution analysis (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 stem cell frequencies of DMSO and merestinib treated samples are shown. Chi-squared, ****p≤0.0001.

Figure 5. Merestinib blocks MNK signaling and improves survival in an intracranial GBM xenograft

A–B, Nude mice were injected with luciferase-expressing U87 cells by intracranial injection. On days 0 and 3 of treatment, bioluminescence imaging (BLI) was performed in vehicle and merestinib treated mice. BLI pictures from one vehicle treated and one merestinib mouse with similar starting values are shown. Graph represents means ± SEM of day 3 BLI values normalized to day 1 values. Unpaired two-tailed t-test, p=0.14. C, Survival analysis of vehicle (n=10) and merestinib (n=11) treated mice. Log-rank (Mantel-Cox) test, p=0.0295. Red arrows indicate 2 treatment cycles (5 days of treatment, 2 days of rest). D, Immunohistochemistry (IHC) staining of brain tumors from vehicle and merestinib treated mice are shown. H&E staining (upper panels) and IHC for phopsho-eIF4E (Ser209) (lower panels) are shown. Scale bar = 50 μm. E, Rank order of IHC for phospho-eIF4E (Ser209) in vehicle and merestinib treated samples is shown. Spearman rank correlation (two-tailed), p=0.046. F, Number of mitoses per 10 high-power fields in brain tumors from vehicle and merestinib treated mice are shown. Unpaired two-tailed t-test, p=0.06.