Methylation-Dependent Tissue Factor Suppression Contributes to the Reduced Malignancy of IDH1 Mutant Gliomas

Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1078-0432.CCR-18-1222 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Unruh et al.

Methylation-dependent Tissue Factor suppression contributes to the reduced malignancy of IDH1 mutant gliomas

1 1 2 1 1 Dusten Unruh, Snezana Mirkov, Brian Wray, Michael Drumm, Jonathan Lamano, Yuping D

Li,1 Qazi F. Haider,1 Rodrigo Javier,1 Kathleen McCortney,1 Amanda Saratsis,1 Denise M.

Scholtens,3 Jann N. Sarkaria,4 C. David James,1 Craig Horbinski1,5

1Department of Neurological Surgery, Northwestern University, Chicago, IL, 60611, USA

2Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL,

60611, USA

3Department of Preventive Medicine, Northwestern University, Chicago, IL, 60611, USA

4Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 55905, USA

5Department of Pathology, Northwestern University, Chicago, IL, 60611, USA

RUNNING TITLE

Tissue Factor in IDH1mut and IDH1wt gliomas

KEYWORDS

Tissue Factor; glioma; isocitrate dehydrogenase; beta-catenin; protease-activated receptor

FINANCIAL SUPPORT

This work was supported by NIH grants K08CA155764 and R01NS102669 (C.H.). D.U. was supported by NIH grants T32CA070085 and F32CA216996. C.H., D.M.S., and C.D.J. were also supported by the Northwestern SPORE in Brain Cancer P50CA221747. The Mayo Clinic Brain

Tumor Patient-Derived Xenograft National Resource is supported by Mayo Clinic, the Mayo

SPORE in Brain Cancer P50CA108961 and NIH grant R24NS92940. Histology and fluorescent

Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1078-0432.CCR-18-1222 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Unruh et al. microscopy services were provided by the Mouse Histology and Phenotyping Laboratory and the Center for Advanced Microscopy/Nikon Imaging Center, respectively; both are supported by

NCI P30CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center.

CORRESPONDING AUTHOR

Craig Horbinski, M.D., Ph.D.,

Northwestern University

Tarry 2-705

300 East Superior St.

Chicago, IL 60611

Phone: 312-503-6127

Email: [email protected]

CONFLICTS OF INTEREST

The authors disclose no potential conflicts of interest

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TRANSLATIONAL RELEVANCE

IDH1mut gliomas are much less aggressive than IDH1wt gliomas, though it is still not fully understood why. We recently discovered that IDH1mut gliomas produce far less TF than IDH1wt gliomas. TF is well known for triggering blood coagulation, but it also promotes malignant behavior via activation of protease-activated receptor 2 (PAR2). Using patient-derived IDH1wt and IDH1mut glioma xenografts, this work (i) defines a mechanism for TF suppression in IDH1mut gliomas; (ii) advances our understanding of how TF-PAR2 signaling contributes to glioma malignancy; (iii) describes a novel mechanism for cancer resistance to receptor tyrosine kinase inhibitors; (iv) sheds light on why it is so difficult to develop patient-derived models of IDH1mut gliomas; (v) identifies a new therapeutic target for treating IDH1wt gliomas.

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ABSTRACT

Purpose: Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in IDH1mut glioma patients. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas.

Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways.

Results: A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P=0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P=0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTKs) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of β-catenin.

Conclusions: These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.

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INTRODUCTION Approximately 20-30% of diffusely infiltrative gliomas contain point mutations in the metabolic enzyme isocitrate dehydrogenase 1 or, less commonly, IDH2 (hereafter collectively referred to as “IDH1mut”) (1). These mutations unmask latent oxidoreductase activities, causing

α-ketoglutarate to be converted into D-2-hydroxyglutarate (D2HG) (2). The D2HG product of

IDH1mut competitively inhibits many α-ketoglutarate-dependent DNA and histone demethylases, resulting in genomic hypermethylation and altered transcription of many genes (3,4). Although

IDH1mut has been linked to suppression of cellular differentiation and predisposition toward oncogenic transformation, it is also associated with lower World Health Organization (WHO) grade and longer survival in glioma patients (5-7). As further indication of their attenuated malignancy, IDH1mut gliomas are much more difficult to grow in culture or as patient-derived xenografts (PDX), relative to IDH1wt gliomas (8,9). The basis for the lesser malignancy of

IDH1mut tumors is far from fully understood.

Previously, we reported that the absence of intratumoral microthrombi is the best histologic predictor of IDH1mut in gliomas, independent of WHO grade and patient age (10). We also found that IDH1mut gliomas are far less likely to cause systemic venous thromboemboli

(VTE) than IDH1wt gliomas, again independent of WHO grade and patient age. Further investigation into the mechanistic underpinnings for these clinically-based observations revealed that F3, the gene encoding Tissue Factor (TF), is one of the most hypermethylated and downregulated genes in IDH1mut gliomas (10). TF is a highly conserved glycoprotein that binds with activated Coagulation Factor VII (FVIIa) to initiate the clotting cascade, and its production and release by cancers into the systemic circulation have been repeatedly linked to VTE in patients (11). Circulating TF is lower in patients with IDH1mut gliomas than IDH1wt gliomas, and elevated plasma TF positively correlates with increased risk of glioma-induced VTE (10).

In addition to its procoagulant activity, the TF-FVIIa complex can also activate transmembrane G protein-coupled protease-activated receptors (PARs), especially PAR2 (12).

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TF-PAR2 activity is important for wound healing at sites of vascular injury and hemostasis. In cancer, however, TF-PAR2 initiates signaling cascades that promote growth, angiogenesis, invasion, and metastasis (13). An unbiased screen for metastatic enhancer elements found that

TF was a major contributor to tumor metastasis (14). Less is known about the activity of TF in gliomas, although it has been shown to increase malignant phenotypes when ectopically expressed in glioblastoma (GBM) cell lines (15-17). Our prior work showed that, in patients with

IDH1wt GBM, high intratumoral TF protein expression correlated with an approximately 50% reduction in median survival (10). Therefore, we hypothesized that TF enhances the malignancy of IDH1wt gliomas, and that its suppression is an important component of the less aggressive

IDH1mut glioma phenotype.

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MATERIALS AND METHODS

Cell Lines and Cell Culture

Three patient-derived glioma cell types with endogenous IDH1mut were used: GBM164 from

Mayo Clinic; anaplastic oligodendroglioma BT142 from American Type Culture Collection

(ATCC) (18); anaplastic astrocytoma TB09 from Dr. Hai Yan at Duke University (19). All three cell types express R132H IDH1 (Fig. 1C); to ensure retention of functional IDH1mut, cells were regularly subjected to Sanger sequencing analysis, and their ability to produce D2HG was verified by liquid chromatography-mass spectrometry (LC-MS) (Fig. S1B and S1C). BT142 cell line lost the IDH1wt allele, and therefore no longer produces high levels of D2HG (Fig. S1C).

GBM164 was maintained as slow-growing subcutaneous flank PDX, with short intervals of in vitro cultures supplemented with 110 mg/mL pyruvate. TB09 was cultured in 1:1 ratio of DMEM

(Corning) and NeuroCult NS-A (STEMCELL Technologies) with 10% FBS, 1% penicillin streptomycin (PS), 2 µg/mL heparin sulfate, 10 ng/mL fibroblast growth factor, 4.5 g/L sodium pyruvate, and 20 ng/mL epidermal growth factor. IDH1wt gliomas (GBM6, GBM12, GBM43) were maintained as PDX or cultured briefly for in vitro testing and have been described elsewhere

(20,21). IDH1wt gliomas were cultured in DMEM supplemented with 10% FBS and 1% PS. The unique identity of all 6 cell lines was authenticated using short tandem repeat analysis (Table

S1), and cells were periodically verified to be mycoplasma-free with a testing kit (Sigma Aldrich).

Knockdown and Overexpression Studies

F3-specific short hairpin RNA (shRNA) sequences were designed and cloned into

BLOCK-iT U6 RNAi Entry Vector (Invitrogen) (Table S2). F3 shRNA or empty vector control cassettes were then recombined into a BLOCK-iT Viral RNAi vector (Invitrogen), and used to generate lentiviral particles for stable constitutive shRNA delivery.

F3 cDNA was cloned into pcDNA4/TO/myc-His Vector (Invitrogen) under control of CMV promoter. F3 cDNA cassettes were then transfected into cells using Lipofectamine 2000

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(Thermo Fisher Scientific). For generation of stable transfectants, cells were selected with zeocin.

Matrigel Chemoinvasion Assay

Cell invasiveness was assessed using polycarbonate membrane transwell inserts with 8.0 µm pores (Corning). Cells were serum starved for 24 hours prior to Matrigel chemoinvasion assay.

Transwell membranes were coated with a thin layer Matrigel basement membrane matrix

(Corning). Culture medium containing 10% FBS was used in the lower chamber as chemoattractant. 1x105 cells were added to the upper chamber in serum-free medium and allowed to migrate for 12 hours. Membranes were fixed with cold methanol, stained with

Vectashield mounting medium containing DAPI (Vector Laboratories), and a cotton swab was used to remove non-migrated cells from the upper chamber. Migrated cells were counted in 3-5 fields per membrane and performed in triplicates.

Soft-Agar Anchorage Independent Growth Assay

Cellular anchorage-independent growth was measured by preparing two layers of agar (BD

Bioscience). The bottom layer of agar was prepared at 1%, autoclaved, and diluted 1:1 with 2x cell culture medium; 1.5 mL was added to each well of a 6-well tissue culture plate and allowed to solidify. The top layer of agar was prepared at a 0.6% agar concentration, and mixed with

5,000 cells per well. Plates were cultured at 37°C in 5% CO2 and fed twice a week. After 3-5 weeks, the colonies were stained with nitroblue tetrazolium chloride (Sigma Aldrich), imaged using BioRad ChemiDoc MP Imaging System, and quantified with ImageJ.

Proteome Profiler Phospho-Kinase Array

The proteome profiler human phospho-kinase Array Kit (R&D Systems) was used according to the manufacturer’s protocol. Briefly, membranes were incubated with blocking buffer for 1 hour,

Unruh et al. then incubated with cell lysates at a protein concentration of 400 µg/membrane overnight at

4°C. After incubation, membranes were washed, incubated with detection antibody cocktails for

2 hours at room temperature, re-washed, and incubated with Streptavidin-HRP solution for 30 min at room temperature. The membranes were then washed again, incubated with Chemi

Reagent Mix, imaged with BioRad ChemiDoc MP Imaging System, and quantified with ImageJ.

In vivo Studies

Nude mice (NCr-Foxn1nu) were purchased from Taconic. All mice were housed under an aseptic barrier facility and handled in compliance with the Institutional Animal Care and Utilization

Committee, Northwestern University. Gliomas (2x105 cells) were implanted into the right frontal lobes of mice aged 5-6 weeks. Engrafted mice were imaged 1-2 times weekly by bioluminescence imaging (BLI). BLI was performed by subcutaneous injection with 150 mg/kg

D-luciferin potassium (Gold Biotechnology) in mice anesthetized via 2.5% isoflurane; each mouse was imaged 10 min after luciferin injection on an IVIS SPECTRUM imaging station.

Overall survival endpoints were determined by neurological symptoms or weight loss that exceeded 20% of original body weight.

Statistical Analyses

Differences between mean values of two groups were compared using two-sample t- test, or between multiple groups by one-way analysis of variance (ANOVA) and post hoc

Tukey’s test; P values less than 0.05 were considered significant. Kaplan-Meier and t-tests were performed to compare survival between groups. Graph generation and statistical analyses were performed with GraphPad PRISM 5 software.

Additional methods can be found in the “Supplementary Methods” section.

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RESULTS

IDH1mut gliomas suppress TF expression via F3 DNA methylation

The 3 major molecular subdivisions of diffusely infiltrative gliomas are, in order of increasing malignancy: (i) IDH1mut with 1p/19q codeletion (oligodendrogliomas); (ii) IDH1mut with intact 1p/19q (astrocytomas); (iii) IDH1wt (GBM) (22). In an analysis of 656 WHO grade II-IV gliomas in The Cancer Genome Atlas (TCGA) via GlioVis (23), F3 mRNA showed a strong inverse relationship with IDH1mut in both low-grade (II-III) and high-grade (IV) gliomas (P<0.001 in both grade II-III and grade IV tumors) (Fig. 1A). On average, IDH1mut gliomas with 1p/19q codeletion expressed slightly less F3 mRNA than IDH1mut gliomas with intact 1p/19q (Fig. 1A), and also produced less mRNA from the gene encoding the major TF receptor, PAR2 (F2RL1), than either IDH1wt gliomas or IDH1mut gliomas with intact 1p/19q (Fig. S1A). In patient-derived glioma cells with endogenous IDH1mut, F3 mRNA and TF protein levels were both markedly lower than in patient-derived IDH1wt glioma cells (Fig. 1B and 1C), whereas PAR2 was present in all cells (Fig. 1C). All 3 IDH1mut cell types expressed R132H IDH1 (Fig. 1C).

F3 contains a methylation-sensitive CpG island in its promoter region (24). F3 was markedly hypermethylated in IDH1mut glioma cells (TB09, BT142, GBM164) relative to IDH1wt glioma cells (GBM6, GBM12, GBM43) (Fig. 1D), consistent with our prior analysis of TCGA gliomas (10). F3 methylation was highest in the 3’ end of the CpG island, as well as in CpG sites ≥2 kb 3’ to the CpG island, termed the “north CpG island shore.” This area is critical for the methylation-dependent suppression of many genes in cancers (25). To establish a direct causative link between IDH1mut and F3 methylation, we analyzed publicly available data from human neural stem cells engineered to express IDH1mut (GEO: GSE94962) (26), and found the same pattern of induced F3 hypermethylation and F3 mRNA reduction as in our IDH1mut cells and TCGA gliomas (Fig. S1D and S1E). Treatment with a demethylating agent, decitabine

(DAC), caused a 5.4-fold increase in F3 mRNA in IDH1mut glioma cells (P=0.018), but had no effect on F3 mRNA in IDH1wt glioma cells (P=0.35) (Fig. 1E and 1F). DAC also increased the

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TF procoagulant activity (PCA) of IDH1mut glioma cells by 2.3- to 19.9-fold, but not of IDH1wt glioma cells (Fig. S1F).

It was recently shown that alteration of the 1:1 IDH1wt:IDH1mut allele ratio, with subsequent reduction in global CpG methylation, develops in a subset of IDH1mut gliomas during recurrence and malignant progression (27). Using the publicly accessible data from that study, we analyzed F3 methylation in matched original and recurrent IDH1mut gliomas (European

Genome-Phenome Archive database accession #EGAD00010001408). Of the 3 patients whose

IDH1mut gliomas acquired an allelic imbalance on recurrence, 2 had reduced tumor F3 methylation in the north shore and CpG island, whereas recurrent IDH1mut tumors that maintained a 1:1 IDH1wt:IDH1mut ratio showed little change in F3 methylation (Fig. S2A).

IDH1mut is associated with significantly better survival in gliomas, but not in other cancers that often contain these mutations, including acute myeloid leukemia (AML) (28), chondrosarcoma (29), melanoma (30), and cholangiocarcinoma (31). In comparison with these other types of IDH1mut cancer, IDH1mut gliomas showed a 5-fold median increase in F3 methylation (average F3 CpG β-value increase=0.31 in IDH1mut gliomas versus 0.06 in all other

IDH1mut cancers) (Fig. S2B). Likewise, IDH1mut gliomas showed an 18.7% decrease in F3

wt mut mRNA log2 compared to IDH1 gliomas, whereas the other IDH1 tumors only showed an average 6.1% decrease relative to their respective IDH1wt counterparts (Fig. S2C). Together, these experimental and in silico results suggest that TF is suppressed in IDH1mut gliomas through CpG methylation, and that high F3 methylation and TF suppression are relatively unique to IDH1mut gliomas.

Ectopic expression of TF increases the malignant behavior of IDH1mut gliomas

TF-initiated signal transduction is known to enhance the aggressiveness of many cancers. Ectopic expression of TF via F3 cDNA transduction in IDH1mut cells caused an increase in ERK1/2 and Akt activation, the latter being especially prominent in GBM164 cells

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(Fig. 2A and 2B). TF overexpression in IDH1mut cells increased cell proliferation by 30% and

140% in TB09 and GBM164 cells, respectively (Fig. 2C). TF also increased IDH1mut cell invasiveness (TB09=2.7-fold, P<0.01; GBM164=8.8-fold, P<0.001) (Fig. 2D and 2E) and anchorage-independent growth (TB09=2.4-fold, P<0.001; GBM164=2.6-fold, P<0.001) (Fig. 2F and 2G).

As is typical for most IDH1mut gliomas, neither unmodified TB09 nor unmodified GBM164 cells grow as intracranial PDX in athymic mice. F3 transduction and associated ectopic expression of TF greatly increased intracranial engraftment and growth of GBM164 cells, as indicated by BLI (0/10 control versus 10/10 TF+, P=0.0007, Fisher’s exact test) (Fig. 2H and 2I;

Fig. S3A). However, F3 cDNA had no effect on TB09 cell intracranial engraftment and growth

(not shown). Intracranial growth of F3-modified GBM164 was initially slow, with a sharp increase in growth occurring after 3 months, resulting in a median survival of 6 months for animal subjects post engraftment, versus no deaths (and no tumor detected microscopically, not shown) in control GBM164 mice at day 180 (P=0.02) (Fig. 2I and 2J). Furthermore, while unmodified GBM164 can grow in the mouse flank, F3 cDNA greatly enhanced its engraftment frequency, growth rate, and subcutaneous spread (Fig. S3B-S3D).

TF suppression decreases the malignant behavior of IDH1wt gliomas

We next examined the effect of TF knockdown in patient derived IDH1wt glioma cells that express high levels of TF (Fig. 1B and 1C). GBM6 has amplification of the constitutively active

EGFRvIII (EGFRvIIIamp), GBM12 has EGFRwt amplification, and GBM43 has an inactivating NF1 mutation (21,32). Two different F3-targeted shRNA suppressed TF expression following tumor cell transduction, with construct #2 showing greatest suppression (Fig. 3A and 3B); F3 shRNA

#2 was therefore used in all subsequent experiments. Knocking down TF reduced ERK1/2 and

Akt phosphorylation in EGFR-driven GBM6 and GBM12 cells, but had negligible effect on pERK1/2 and pAkt in NF1-driven GBM43 cells (Fig. 3B). TF knockdown reduced cell

Unruh et al. proliferation by 48.9% in GBM6 and by 55.7% in GBM12, but had no significant effect on

GBM43 proliferation (Fig. 3C). However, the invasiveness of all 3 IDH1wt cell types was reduced by 32-47% (P<0.001) (Fig. 3D). TF knockdown also reduced anchorage-independent growth by

2.3-fold and 14.6-fold in GBM6 and GBM12 cells, respectively (P<0.001), whereas GBM43 cells were unable to form colonies in soft agar, even under control conditions (Fig. 3E).

TF knockdown markedly impaired the intracranial growth of IDH1wt, EGFR-driven GBM6 and GBM12 PDX. The median survival of mice bearing GBM6 was 85% longer when TF was suppressed (77.0 versus 41.5 days, P=0.001) (Fig. 3F). The effect of TF suppression was even more striking in GBM12 PDX, decreasing the bioluminescence of intracranially injected cells to undetectable levels (Fig. 3G). None of the F3 shRNA GBM12 mice died at 90 days, whereas the median survival for mice bearing GBM12 vector control (vc) shRNA was 41.0 days post engraftment, and all were dead before 60 days (P<0.001) (Fig. 3G). Microscopic analysis at 90 days found no detectable tumors in mice injected with GBM12 expressing F3 shRNA (Fig.

S3E). However, F3 shRNA had no effect on tumor growth or median survival of mice engrafted with the NF1mut GBM43 PDX (Fig. 3H). To determine whether the malignant behavior of F3 shRNA-transduced cells could be rescued, knockdown cells were transduced with F3 cDNA- encoding lentivirus resulting in elevated TF expression (F3 rescue) (Fig. 3I). Interestingly, F3 rescue increased tumor growth beyond that of corresponding unmodified parental cells, even in

GBM43 (Fig. S3F, S3G; Fig. 3F, 3G, 3H).

We previously demonstrated that low TF immunostaining correlated with significantly longer overall survival in IDH1wt GBM patients (10). Consistent with this, analysis of F3 and survival in IDH1wt TCGA GBM patients using Cutoff Finder (33) showed that low F3 mRNA correlated with 55% longer median overall survival (20.1 versus 13.0 months, HR=0.58, 95%

CI=0.47-0.73, P<0.0001) (Fig. 3J). Low F3 mRNA was also a significant favorable prognostic marker for overall survival in TCGA GBMs (HR=0.65, 95% CI=0.47-0.91, P=0.011), independent of IDH1mut and patient age (Table S4). An analysis of 10,844 PANCAN TCGA

Unruh et al. cancers showed a similar relationship between lower F3 mRNA and longer survival (7.9 years versus 5.3 years, HR=1.7, 95% CI=1.6-1.9, P<0.0001) (Fig. S3H).

Signaling pathways affected by TF modulation in IDH1mut and IDH1wt gliomas

We next investigated the effects of TF/F3 manipulation in IDHmut and IDHwt gliomas using a human phospho-kinase antibody array. In IDH1mut TB09 cells, F3 transduction and expression increased cellular β-catenin, as well as phosphorylation of PRAS40, JNK1/2, and MSK1, and decreased phosphorylation of STAT3, p53, HSP60, and WNK1 (Fig. 4A). In IDH1mut GBM164 cells, TF overexpression increased production of β-catenin and phosphorylation of ERK1/2,

HSP60, and Akt1/2/3 (Fig. 4B). TF knockdown in IDH1wt GBM6 and GBM12 cells lowered expression of β-catenin and reduced phosphorylation of EGFR, ERK1/2, and Akt1/2/3 (Fig. 4C and 4D), whereas IDH1wt GBM43 cells only showed lower β-catenin expression and reduced

Wnk1 phosphorylation (Fig. 4E).

In a reverse phase protein array (RPPA) analysis of TCGA gliomas, high F3 mRNA

(defined by the 75th percentile) correlated with increased expression of proteins, and the phosphorylation of proteins, involved with EGFR (q<0.0001), mTOR (q<0.0001), and extracellular matrix-receptor (q<0.05) KEGG signaling pathways, as indicated by pathway enrichment analysis (Fig. 4F). Gliomas with high F3 also showed biological process enrichment for proteins involved in cell migration (q<0.001), inhibition of anoikis (q<0.05), and cell survival

(q<0.001) (Fig. 4F).

TF-mediated stimulation of -catenin expression, and the effect of -catenin on glioma cell migration

Among IDH1mut and IDH1wt cells studied, the most consistent phenotypic and molecular changes resulting from TF modulation were altered invasiveness and β-catenin expression. In an analysis of 670 WHO grade II-IV TCGA gliomas, F3 mRNA strongly correlated with β-catenin

Unruh et al. mRNA, CTNNB1 (P<0.001; R2=0.098) (Fig. 5A). Since β-catenin is known to promote invasion in many cancers, including glioma (34), we hypothesized that the pro-invasive effects of TF are mediated predominately through β-catenin. When unmodified IDH1wt cells were cultured in serum-free conditions (to eliminate any serum-based TF and FVIIa), β-catenin protein expression increased and peaked at 2-8 hours after treatment with exogenous recombinant TF

(recTF) and FVIIa (Fig. 5B). Treatment of IDH1wt cells for 24 hours with β-catenin inhibitor XAV-

939 (35) was only able to partially inhibit cell proliferation in GBM6 and GBM12 at 100 µM, and had no effect on GBM43 proliferation (Fig. 5C). Treatment of IDH1wt cells with XAV-939 decreased cell viability at the highest concentration for all cells by 29-53% (P<0.001), with

GBM6 cells showing slightly more sensitivity (Fig. 5D). XAV-939 treatment increased cell apoptosis of GBM6 at 10 µM and 100 µM by 7.2% and 12.6% respectively but did not affect apoptosis in GBM12 or GBM43 cells (Fig. 5E). In contrast, XAV-939 was much more potent against chemoinvasion in all 3 IDH1wt cell types (Fig. 5F and 5G). These findings suggest that the pro-invasive effect of TF in gliomas is dependent on β-catenin.

Tissue Factor promotes ligand-independent transactivation of receptor tyrosine kinases

RTKs, such as EGFR, are well known to stimulate downstream TF production (36,37).

However, our phospho-kinase data indicated that TF may also, in turn, contribute to RTK activation (Fig. 4). In serum-free media, exogenous recTF/FVIIa increased pEGFR, as well as downstream pAkt and pERK1/2, in EGFRvIIIamp GBM6 and EGFRamp GBM12 cells (Fig. 6A). In

NF1mut GBM43 cells, recTF/FVIIa had no effect on pEGFR, a weaker effect on pAkt, and a less durable effect on pERK1/2, especially in comparison with GBM12 (Fig. 6A). The TF/FVIIa complex causes activation of PAR2 and, to a lesser degree, PAR1, PAR3, and PAR4 (38). Of the four major PAR genes, IDH1wt glioma cells express mainly F2RL1, encoding PAR2 (Fig.

S4A). A PAR2-specific activating peptide (PAR2-AP) modestly increased cell proliferation and chemoinvasion in GBM6 and GBM43 cells, but only increased chemoinvasion in GBM12 cells

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(Fig. S4B and S4C). PAR2-AP triggered EGFR, Akt, and ERK1/2 phosphorylation to a much greater extent in GBM6 and GBM12 than in GBM43 (Fig. S4D). A pharmacologic PAR2 inhibitor, GB83, reduced cell proliferation and chemoinvasion in all 3 IDH1wt cells (Fig. S4F and

S4G).

G protein-coupled receptors (GPCRs), like PAR2, can transactivate RTKs through autocrine/paracrine mechanisms that involve extracellular release of RTK ligands, or by intracellular, ligand-independent mechanisms involving kinases such as Src (39). To determine whether TF transactivates EGFR through PAR2 in gliomas, the effect of recTF/FVIIa on EGFR kinase activity and phosphorylation was tested, in conjunction with the following specific pharmacologic inhibitors: (i) cetuximab, an extracellular inhibitor of EGFR activation; (ii)

AG1478, which inhibits intracellular EGFR phosphorylation; (iii) PAR2 antagonist GB83; (iv) Src antagonist SKI-606. Both cetuximab and AG1478 blocked the ability of EGF to activate EGFR, whereas GB83 did not (Fig. 6B). In contrast, both AG1478 and GB83 blocked the effect of recTF/FVIIa on EGFR, but cetuximab did not (Fig. 6B). SKI-606 reduced recTF/FVIIa-induced

EGFR activation (Fig. 6C), and only AG1478, not cetuximab, was able to block the enhancing activity of TF/FVIIa on proliferation (Fig. 6D). These results suggest that TF-PAR2 transactivates EGFR through cytoplasmic domain interactions that involve Src, even when an extracellular EGFR inhibitor is present.

Among IDH1wt gliomas, the extent to which TF-PAR2 activated EGFR correlated with the effect of TF knockdown on in vitro proliferation and in vivo growth (Fig. 3 and 6, and Fig. S3).

F3 cDNA promoted cell proliferation and PDX growth of IDH1mut GBM164 cells, while having little effect on these properties in IDH1mut TB09 cells (Fig. 2 and Fig. S3). We hypothesized that the differential effect of TF on GBM164 versus TB09 may be due to GBM164 expressing one or more RTKs that TB09 does not. On phospho-kinase immunoblots, both TB09 and GBM164 cells showed low basal pEGFR, which did not increase in response to F3 cDNA (Fig. 4A and

4B). Other major RTKs involved in glioma malignancy, including platelet-derived growth factor

Unruh et al. receptor alpha (PDGFRα), PDGFRβ, and Met, were extremely low or undetectable in TB09 cells

(Fig. 6E). In contrast, PDGFRβ was readily evident in GBM164, and was phosphorylated after

TF induction (Fig. 6E). Treatment with exogenous recTF/FVIIa also triggered PDGFRβ phosphorylation in GBM164, with corresponding increases in pAkt and pERK1/2 (Fig. 6F).

Treating TF-expressing GBM164 cells with the PDGFRβ inhibitor (sunitinib malate) prior to implantation blocked in vivo growth (Fig. S3A), although pretreatment with PDGF-BB ligand was unable to facilitate the growth of GBM164 control cells (not shown). We then sought to determine whether PDGFRβ expression in TB09 cells modified to express TF would increase cell proliferation and/or in vivo growth (Fig. 6G). TB09 cells expressing both TF and PDGFRβ had increased pPDGFRβ (Fig. 6G) and greater proliferation than either in isolation (Fig. 6H), but were still unable to grow in vivo (not shown). Together, these data suggest that TF-PAR2 is capable of intracellular RTK transactivation, and that the effect of TF on cell proliferation and tumorigenesis may depend in part on the extent to which RTKs are expressed and activated.

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DISCUSSION

The existence of “Tissue Factor” has been known since the 19th century, when it was discovered that an unknown factor, present in homogenized extracts from animal tissues, could trigger blood clotting (40). TF is a highly conserved protein, found even in ancient invertebrates like the horseshoe crab, and plays a critical role not just in coagulation, but also in immunity and wound healing (41). High TF activity is common in many cancers, and enhances cell proliferation, invasion, and metastasis. Our data indicate that its consistent suppression in

IDH1mut gliomas contributes to the less aggressive behavior seen in this unique subset of tumors, and that blocking TF signaling could prove effective in suppressing the malignant phenotypes of IDH1wt gliomas.

IDH1mut leads to global hypermethylation, a common finding among all IDH1mut cancers.

However, IDH1mut is only associated with reduced malignancy in gliomas, for reasons that remain unclear. One clue is that the targets of IDH1mut-related hypermethylation vary greatly depending on the cellular and microenvironmental contexts in which the mutation occurs (42).

Strong, consistent hypermethylation and downregulation of F3 appears to be a distinct feature of IDH1mut gliomas relative to other IDH1mut tumors (Fig. S2B and S2C). Because of events like

F3 suppression, IDH1mut may indirectly impose limits on glioma malignancy—limits that gradually disappear as the tumor evolves beyond the need for functional IDH1mut and D2HG

(Fig. S2A) (27). Demethylating agents like DAC may therefore have undesirable side effects in

IDH1mut gliomas. In this study, we found that treating IDH1mut TB09 with DAC increased TF levels to that observed for high TF-producing IDH1wt GBM12 (Fig. S1F) (of note, the patient from which GBM12 was derived died of pulmonary embolism (20)). However, DAC still resulted in a net decrease in cell proliferation (Fig. S1G). This may be due to its relatively broad mechanism of molecular action, wherein it not only demethylates DNA, but also results in stalled replication forks and the induction of DNA damage (43,44). F3 methylation often diminishes in

IDH1mut gliomas when they lose the normal 1:1 IDH1wt:IDH1mut allele ratio and progress in

Unruh et al. malignancy (Fig. S2A). In a patient with paired pre-therapy and postmortem glioma samples, the loss of IDH1mut correlated with markedly increased TF expression and hypercoagulation

(Fig. S5).

When TF complexes with FVIIa, it can either activate cell-free Factor X in the clotting cascade, or it can activate PAR2 on cell surfaces by cleaving PAR2’s extracellular tail, leading to intracellular activation of associated G proteins. Under normal circumstances, PAR2 activation stimulates processes involved in wound healing, like cell proliferation, migration, and angiogenesis, but cancers use it to enhance growth and metastasis (13). Indeed, this is one reason why cancer is often referred to as “the wound that does not heal” (45). Prior work by others showed that suppression of TF forces U373 glioma cells into a dormant, undetectable state in vivo for up to a full year, followed by rapid tumor growth after TF restoration (16). Our data indicate that the methylation-dependent suppression of TF in IDH1mut gliomas could help explain why IDH1mut gliomas are less malignant, and have proven difficult to culture and engraft.

Along those lines, it would also be of interest to “rescue” F3/TF expression in nonneoplastic progenitor cells engineered to overexpress IDH1mut (Fig. S1D and S1E), and observe the effects on cell behavior. Another approach would be to induce IDH1mut in an established IDH1wt cancer cell line. However, as such a cell model is inherently more artificial, it may not have the same biologic relevance as direct manipulation of actual patient-derived IDH1mut and IDH1wt glioma cells (46).

In the present study, modification of TF expression in IDH1mut and IDH1wt cells led to changes in multiple biological processes, including cell migration, adhesion, and proliferation.

TF knockdown in IDH1wt gliomas reduced in vitro proliferation and in vivo growth, but only in cells driven by EGFR. TF knockdown completely prevented the in vivo growth of GBM12, in which amplified EGFRwt depends on interaction with other proteins for its activation, and TF knockdown delayed but did not prevent growth of EGFRvIIIamp GBM6, which retains some

EGFR activity even when TF is suppressed (Fig. 3 and Fig. 6A). In contrast, GBM43, in which

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NF1mut exerts pro-cancer effects downstream of EGFR, was completely resistant to the anti- growth effects of TF suppression. However, F3 cDNA still managed to enhance GBM43 growth

(Fig. 3H) and pAKT and pERK1/2 (Fig. 6A); this may be explained by the differences in RTKs expressed by the three IDH1wt glioma cells (Fig. S4E), whereas GBM43 does not express

EGFR it does express PDGFRβ, a RTK that TF is capable of phosphorylating. Furthermore, pharmacologic PAR2 inhibition by GB83 inhibited GBM43 cell proliferation (Fig. S4F), suggesting that either GBM43 cells still benefit from PAR2 activation through mechanisms independent of EGFR, or that GB83 has off-target effects.

TF has previously been reported to be a downstream effector of EGFR and participates in EGFR signaling (37,47). But to our knowledge, the current study is the first to provide detailed evidence that it acts as an upstream activator of EGFR and other RTKs through PAR2, even increasing EGFRvIII phosphorylation beyond its constitutively active baseline (Fig. 4 and 6). Of potential clinical relevance is the discovery that TF does this via purely intracellular mediators, and can activate EGFR even in the presence of cetuximab (Fig. 6B). Cetuximab is clinically effective against RTK-driven carcinomas arising in the large intestine and head and neck (48), but it is still not curative, and transactivation of EGFR by TF-PAR2 is a potential mechanism of therapeutic resistance—one that warrants further investigation in those cancers. Our hypothesis that TF is dependent on RTKs to promote proliferation and tumorigenesis is also consistent with the IDH1mut data, in which PDGFRβ-expressing GBM164 cells benefited much more from TF induction compared to RTK-deficient TB09 cells, in terms of enhanced proliferation and in vivo growth (Fig. 2 and 6). These novel TF-PAR2-RTK activity relationships therefore suggest potential combination therapies for treating IDH1wt gliomas.

In the current study, the most consistent pathway and phenotypic changes in response to TF modulation among all cells was invasiveness, with indication of elevated β-catenin expression as a key downstream effector of TF-stimulated invasion (Fig. 2-5). The pro-invasive activity of TF appears to be initiated by and dependent on TF interaction with PAR2, as PAR2-

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AP and GB83 increased and decreased invasion, respectively (Fig. S4C and S4F). Activated

PAR2 is known to stabilize β-catenin (49), and β-catenin, in turn, is known to facilitate cell–cell adhesion and cell–matrix adhesion during cancer cell invasion (50). Consistent with this, analysis of TCGA gene expression data revealed that ECM-receptor interactions positively correlate with glioma F3 mRNA (Fig. 4F). Thus, the pro-growth effects of TF may depend primarily on RTK interactions, whereas the pro-invasive effects of TF appear to be predominately mediated through β-catenin.

In sum, these data demonstrate that the activation of PAR2 by the TF/FVIIa complex has two major consequences in IDH1wt gliomas: (i) promotion of cell proliferation and tumor formation through intracellular stimulation of RTK; (ii) β-catenin-dependent upregulation of invasiveness (Fig. S6). Because F3 hypermethylation occurs to a much greater extent in

IDH1mut gliomas than in other IDH1mut cancers, and IDH1mut has only been consistently shown to be a favorable prognostic marker in gliomas, not other cancer types, TF suppression may be central to the lesser malignancy of IDH1mut gliomas. Clinically, this work has multiple implications, including: (i) counteracting IDH1mut activity in gliomas, either directly by enzyme inhibition or indirectly through demethylating drugs, might paradoxically increase tumor aggressiveness and VTE risk; (ii) TF-PAR2 is a potential mechanism of resistance to extracellular EGFR inhibitors currently in use against other cancers; (iii) PAR2 may be an effective anticancer target, without disrupting the normal hemostatic activity of TF.

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ACKNOWLEDGMENTS

We thank Dr. Hai Yan for the TB09 cells, Lisa Magnusson for her assistance with the glioma

PDX models, and Alicia Steffens for immunohistochemical and histochemical staining.

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FIGURES LEGENDS

Figure 1: TF/F3 expression and methylation in IDH1mut and IDH1wt glioma cells. (A) F3 mRNA expression in LGG and GBM TCGA datasets. Wild-type and mutant IDH1 gliomas were compared according to LGG or GBM, with LGG IDH1mut sorted by 1p/19q codeletion. Bars show median with interquartile range. (B) F3 mRNA expression by quantitative RT-PCR (n=3), and

(C) western blot for TF, PAR2, and R132H IDH1 (IDH1mut) protein expression in patient-derived glioma cells that are IDH1wt (GBM6, GBM12, GBM43) or IDH1mut (TB09, BT142, GBM164). (D)

Mean increase in methylation beta values at each CpG site associated with F3, in IDH1mut cells

(TB09, BT142, GBM164) relative to IDH1wt cells (GBM6, GBM12, GBM43). The orange bar at the top indicates CpG sites that reside within the F3 coding region. CpG sites are shown 5’ to 3’ left to right. (E) F3 mRNA expression after 3 days of DAC in IDH1wt GBM6 cells. (F) F3 mRNA expression after 3 days of DAC in IDH1mut TB09 cells. Bar graphs show the mean ± SD of ≥ 3 replicates per condition, P values determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P <

0.001.

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Figure 2: Effect of TF on in vitro and in vivo behavior of IDH1mut gliomas. (A) F3 mRNA expression by quantitative RT-PCR (n=3), and (B) western blot for TF, phospho/total-ERK1/2, phospho/total-Akt, and β-Actin in patient-derived IDH1mut cells with and without F3 cDNA. (C)

Cell proliferation by BrdU incorporation after 72 hrs (n=3). (D) Representative immunofluorescent DAPI-stained images of Matrigel chemoinvasion assay membranes comparing number of IDH1mut migrated cells±F3 cDNA (yellow false color). (E) Quantification of

Matrigel chemoinvasion assay (n=6). (F) Representative photomicrographs of soft agar colony formation assay. (G) Quantification of soft agar colony formation assay comparing IDH1mut cells±F3 cDNA (n=3). (H) Longitudinal representative BLI of luciferase-labeled IDH1mut GBM164 cells with or without F3 cDNA, and (I) tumor growth curves (n=10/group). (J) Kaplan-Meier curves for overall survival of IDH1mut cells±F3 cDNA (GBM164) (n=10/group). Bar graphs show the mean±SD of ≥3 replicates per condition, P values determined by one-way ANOVA.

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Figure 3: Effect of TF knockdown on in vitro and in vivo tumor behavior of IDH1wt gliomas. (A) F3 mRNA expression by quantitative RT-PCR of IDH1wt cells with either vector control shRNA (vc shRNA) or F3 shRNA (n=3). (B) Western blot of IDH1wt cells with either vc or

F3 shRNA #2 for TF, phospho/total-ERK1/2, phospho/total-Akt, and β-actin. (C) Cell proliferation by BrdU incorporation after 24 hrs (n=3). (D) Representative immunofluorescent

DAPI-stained images (yellow false color), and quantification of Matrigel chemoinvasion assay comparing IDH1wt migrated cells with vc or F3 shRNA (n=6) (E) Representative photomicrographs, and quantification of soft agar colony formation assay comparing IDH1wt cells with vc or F3 shRNA (n=3). (F, G, H) Longitudinal representative bioluminescence imaging

(BLI) of luciferase-labeled IDH1wt cells (GBM6, GBM12, GBM43) expressing vc shRNA, F3 shRNA, or F3 rescue with tumor growth curves and Kaplan-Meier curves for overall survival

(n≥5/group). (I) TF protein expression by western blot. (J) Kaplan-Meier curves of overall survival in IDH1wt GBM patients from TCGA data obtained through GlioVis portal (n=525), and high F3 mRNA expression was 8.09, as determined by Cutoff Finder (n=86 for low F3 and 439 for high F3). Bar graphs show the mean ± SD of ≥3 replicates per condition, P values determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

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Figure 4: Comparative effect of TF on Phospho-kinase activation in IDH1mut and IDH1wt gliomas. (A) Phospho-kinase immunoblot of IDH1mut TB09 and (B) GBM164 cells with or without F3 cDNA, and color-coded fold-change of quantified pixel density to the right. Phospho- kinase immunoblot of IDH1wt GBM6 (C), GBM12 (D), and GBM43 (E) expressing either vc shRNA or F3 shRNA, with color-coded fold-change of quantified pixel density to the right of each immunoblot. Blots were processed with equivalent protein concentrations and exposure times. (F) Analysis of proteomic and phosphoproteomic RPPA data for WHO grade II-IV glioma

TCGA patients (n=670), correlated with high (≥75th percentile) versus low (≤25th percentile) F3 mRNA. Each node in the network is color-coded with RPPA expression as it correlates with F3 mRNA expression (blue=positive correlation). Data is derived from the TCGA LGG and GBM glioma datasets. Functional biological processes and physical interactions were identified using

STRING v10.5 and Cytoscape v3.6.0. Biological processes and KEGG signaling pathways were determined with Metascape. Teal boxes represent gene enrichment for biological processes, and green boxes represent KEGG signaling pathways. Significant q-values represent p-values that have been adjusted for the False Discovery Rate. Bar graphs show the mean ± SD, P values determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

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Figure 5: TF-mediated β-catenin signaling and invasiveness in IDH1wt glioma cells. (A)

Scatter plot shows correlation between β-catenin, CTNNB1 mRNA and TF, F3 mRNA. Red line represents linear fit, R2 is Pearson’s correlation coefficient, and P indicates significance determined by two-tailed Student’s T-test. (B) IDH1wt cells treated with increasing concentrations of XAV-939 (0, 1, 10, 100 µM) for 24 hrs and analyzed by western blot and densitometry quantification for β-catenin. (C) Cell proliferation by BrdU incorporation of IDH1wt cells treated with XAV-939 (0, 1, 10, 100 µM) for 24 hrs (n=3). (D) CellTiter-Glo cell viability assay, and (E) Propidium iodide flow cytometry analysis for apoptosis of IDH1wt cells treated with XAV-939 (0, 1, 10, 100 µM) for 24 hrs. (F) Representative immunofluorescent DAPI-stained images of Matrigel chemoinvasion assay membranes comparing of IDH1wt cells treated with

XAV-939 (0, 1, 10, 100 µM) for 24 hrs (yellow false color). (G) Quantification of Matrigel chemoinvasion assay (n=3). Bar graphs show the mean ± SD of ≥ 3 replicates per condition, P values determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

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Figure 6: RTK activation by TF-PAR2 signaling in IDH1wt and IDH1mut gliomas. (A) Western blot of serum-starved IDH1wt cells (GBM6, GBM12, GBM43) stimulated with 50 ng/mL recTF and 10 ng/mL FVIIa (recTF/FVIIa) for 0-120 min and probed for p-EGFR, p-Akt, p-ERK1/2, and

β-actin. (B) Western blot of serum starved GBM12 cells were pretreated with 20 µM GB83, 20

µM AG1478, or 5 µg/mL cetuximab and then stimulated with 20 ng/mL EGF or recTF/FVIIa. (C)

Western blot of serum starved GBM12 cells treated with recTF/FVIIa, and/or 1 µM SKI-606 probed for p-EGFR, p-Src, and Src. (D) Cell proliferation by BrdU incorporation of GBM12 vc and F3 shRNA cells treated with vehicle control, cetuximab, recTF/FVIIa, recTF/FVIIa + cetuximab, or recTF/FVIIa + AG1478 for 24 hrs (n=3). (E) Western blot of IDH1mut cells (TB09,

GBM164) for p-PDGFRβ, PDGFRβ, p-Met, and Met. (F) Western blot of serum starved IDH1mut

(GBM164) cells stimulated with recTF/FVIIa for 0-120 min and probed for p-PDGFRβ, p-Akt, p-

ERK1/2, and β-actin. (G) Western blot of TB09 cells modified to express either F3, PDGFRβ, or both F3 and PDGFRβ cDNA. (H) Cell proliferation by BrdU incorporation of modified TB09 cells expressing TF, PDGFRβ, or both TF and PDGRβ.

Methylation-dependent Tissue Factor suppression contributes to the reduced malignancy of IDH1 mutant gliomas

Dusten Unruh, Snezana Mirkov, Brian Wray, et al.

Clin Cancer Res Published OnlineFirst September 28, 2018.

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