Targeting Resistance Against the MDM2 Inhibitor RG7388 in Glioblastoma Cells by the MEK Inhibitor Trametinib Anne Berberich1,2, Tobias Kessler1,2, Carina M

Published OnlineFirst October 1, 2018; DOI: 10.1158/1078-0432.CCR-18-1580

Research Article Clinical Cancer Research Targeting Resistance against the MDM2 Inhibitor RG7388 in Glioblastoma Cells by the MEK Inhibitor Trametinib Anne Berberich1,2, Tobias Kessler1,2, Carina M. Thome1, Stefan Pusch3,4, Thomas Hielscher5, Felix Sahm4, Iris Oezen6, Lara-Marie Schmitt1,2, Sara Ciprut1, Nanina Hucke1,2, Petra Ruebmann1, Manuel Fischer7, Dieter Lemke1,2, Michael O. Breckwoldt6,7, Andreas von Deimling3,4, Martin Bendszus7, Michael Platten2,6,8, and Wolfgang Wick1,2

Abstract

Purpose: Resistance is an obstacle of glioma therapy. Results: MDM2 inhibition required functional p53 and Despite targeted interventions, tumors harbor primary resis- showed synergistic activity with radiotherapy in first-line tance or become resistant over short course of treatment. This treatment. Long-term exposure to RG7388 induced resis- study examined the mouse double minute 2 (MDM2) inhib- tance by activation of the extracellular signal-regulated itor RG7388 together with radiotherapy and analyzed strate- kinases 1/2 (ERK1/2)–insulin growth factor binding protein gies to overcome acquired MDM2 inhibitor resistance in 1 (IGFBP1) signaling cascade, which was specifically over- glioblastoma. come by ERK1/2 pathway inhibition with trametinib and Experimental Design: Effects of RG7388 and radiothera- knockdown of IGFBP1. Combining trametinib with contin- py were analyzed in p53 wild-type glioblastoma cell lines ued RG7388 treatment enhanced antitumor effects at and glioma-initiating cells. RG7388 resistant cells were RG7388 resistance in vitro and in vivo. generated by increasing RG7388 doses over 3 months. Conclusions: These data provide a rationale for combin- Regulated pathways were investigated by microarray, ing RG7388 and radiotherapy as first-line therapy with a qRT-PCR, and immunoblot analysis and specifically inhib- specific relevance for tumors insensitive to alkylating stan- ited to evaluate rational salvage therapies at RG7388 dard chemotherapy and for the addition of trametinib to resistance. Effects of RG7388 and trametinib treatment were continued RG7388 treatment as salvage therapy after challenged in an orthotopical mouse model with RG7388 acquired resistance against RG7388 for clinical practice. Clin resistant U87MG glioblastoma cells. Cancer Res; 1–13. 2018 AACR.

Introduction inhibitor 2A (CDKN2A; 57.8%), or amplification of mouse double minute homologs 1/2/4 (MDM1/2/4; 15%; ref. 1). Dysregulation of the p53 pathway is found in 85.3% of Overexpression (OE) of MDM2, the key negative regulator of primary glioblastoma and caused by p53 mutation or homo- p53, impairs p53 wild-type function and deregulates the zygous deletion (27.9%), deletion of cyclin-dependent kinase MDM2-p53 feedback loop, which results in an accelerated tumor growth in a variety of human tumors, including sarcoma, leukemia, breast cancer, melanoma, and glioblastoma (2–7). 1Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), 2 Targeting MDM2 evolved as a promising treatment approach to German Cancer Research Center (DKFZ), Heidelberg, Germany. Department of reactivate the p53 pathway (8) leading to cell-cycle arrest, Neurology, Heidelberg University Hospital, Heidelberg, Germany. 3Clinical Cooperation Unit Neuropathology, DKTK, DKFZ, Heidelberg, Germany. increased apoptosis, and decreased tumor growth in human 4Department of Neuropathology, Heidelberg University Hospital, Heidelberg, tumor xenografts in nude mice (9, 10). RG7388, also known as Germany. 5Division of Biostatistics, DKFZ, Heidelberg, Germany. 6Clinical idasanutlin, is a second-generation MDM2 inhibitor of the Cooperation Unit Neuroimmunology and Brain Tumor Immunology, DKTK, nutlin family with superior potency and selectivity compared DKFZ, Heidelberg, Germany. 7Department of Neuroradiology, Heidelberg 8 with its predecessor RG7112 (11). RG7388 binds selectively and University Hospital, Heidelberg, Germany. Department of Neurology, Medical with a high affinity to the p53 binding site on the surface of the Faculty Mannheim, Heidelberg University, Mannheim, Germany. MDM2 molecule by mimicking the three key binding amino Note: Supplementary data for this article are available at Clinical Cancer acids (9, 10, 12) and thereby inhibiting the MDM2-p53 inter- Research Online (http://clincancerres.aacrjournals.org/). action. There are first signs of efficacy for MDM2 inhibitors, Corresponding Author: Wolfgang Wick, University of Heidelberg, INF 400, including RG7388, in studies for patients with leukemia and Heidelberg 69120, Germany. Phone: 00496221567075; E-mail: sarcoma (4, 5, 13). Clinical trials using MDM2 inhibitors for [email protected] patients with glioblastoma have not yet been conducted. doi: 10.1158/1078-0432.CCR-18-1580 Primary and acquired resistance and optimal patient selection 2018 American Association for Cancer Research. are the biggest challenges for the clinical use of targeted therapies.

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Aldrich) using Lipofectamine (Invitrogen) according to the Translational Relevance manufactures protocol. For stable OE with IGFBP1, Gateway The present experiments provide a good rational for a novel compatible IGFBP1 cDNA [NM_000596] and the vector systemic treatment, that is radiotherapy, MDM2, and ERK1/2 pMXS-GW-IRES-PuroR were obtained from the German Cancer inhibition specifically in the so far underserved group of Research Center clone repository. Correct sequence of cDNA patients with glioblastoma with a lack of MGMT promoter was validated by Sanger sequencing (GATC). RG7388 resistant hypermethylation. As is, the combination will be forwarded to cells were generated by twice weekly treatment of U87MG the clinic in a multicenter phase I/II clinical trial with molec- cells with increasing doses of 10 nmol/L to 10 mmol/L RG7388 ular preselection of suitable patients. ("RG7388 resistant cells") or related DMSO amounts ("DMSO control treated cells") over a period of 3 months. Murine cerebellum neurons were freshly isolated from P6 neonatal mice. Cerebelli were digested with typsine/DNAse solution as described more detailed in supplementary meth- For glioblastoma, O6-methylguanine DNA methyltransferase ods. Cells were seeded in poly-L-lysine (PLL) coated 96-well (MGMT) promoter methylation status may be regarded as the microplates at 50,000 cells per well in 200 mL culture media. only predictive biomarker aiding the decision for the use of Treatment was added 24 hours after seeding as indicated in alkylating chemotherapy. However, today's guidelines still advo- respective figures and MTT assay was performed 6 days after cate the use of temozolomide regardless of MGMT status and treatment. fi alternatives for patients not likely bene tting from temozolomide Murine astrocytes were freshly isolated from P1 or P2 neo- – are yet to be developed (14 16). With regards to MDM2 inhibi- natal mice as described previously (27). Cells were seeded in PLL tors, several preclinical studies showed that MDM2 inhibitors coated 96-well microplates at 15,000 cells per well in 200 mL reduced tumor growth in p53 wild-type tumors, whereas tumors complete DMEM media and treated 24 hours later as indicated harboring p53 mutations were primary resistant against the in the respective figures. After another 8 days MTT assay was – fi treatment (17 19). Furthermore, MDM2 ampli cation in p53 performed. wild-type tumors increased sensitivity to MDM2 inhibitory fi treatment strategies highlighting MDM2 ampli cation and p53 Orthotopical xenograft mouse model wild-type status as potential biomarkers for patient selection All animal work was approved by the governmental author- (7, 20). Acquired resistance mechanisms are still not understood, ities (animal application number: G210-16; Regier- especially in glioblastoma. Potential mechanisms leading to ungspr€asidium Karlsruhe) and performed in accordance with acquired MDM2 inhibitor resistance are p53 mutations the German animal protection law. A total of 1 105 RG7388- – (21 24) as well as enhanced B-cell lymphoma-extra-large resistant U87MG cells were stereotactically implanted into the (Bcl-xl) or MDM4 protein expression, offering the opportunity right brain hemisphere of deeply anesthetized CD1 nu/nu mice fi to be targeted by the addition of speci c inhibitors (25). (Charles River Laboratories). Size calculations for the animal In this study, we determined a so far unknown resistance experiment were performed using GPower calculator version mechanism against the MDM2 inhibitor RG7388 in p53 wild- 3.1.9.2 (Universit€at Dusseldorf,€ Dusseldorf,€ Germany) with set- fi type glioblastoma cells, resulting in the identi cation of a new ting of the following variables: a ¼ 0.05, power(b) ¼ 0.80, rational salvage therapy to potentially overcome resistance. estimated standard deviation ¼ 30% of volume. A meaningful biological difference was assumed at 50% reduction in tumor Material and methods volume and the dropout rate due to lack of tumor growth was estimated as 20%. Altogether tumor cell inoculation was carried A more detailed description is given in the supplementary out in 40 animals. MRIs were performed with a 9.4-Tesla hor- methods. izontal-bore small animal MRI scanner (BioSpec 94/20 USR; Bruker BioSpin GmbH) with a four-channel phased-array surface Cell culture receiver coil. A T2-weighted (T2-w) rapid acquisition with relax- The human glioblastoma cell lines U87MG, A172, T98G, ation enhancement (RARE) sequence was used to assess tumor LN428, LN308 (ATCC) and LN229 (N. de Tribolet), were kept volume. Tumor segmentation was performed in Amira (FEI) under standard conditions. The primary glioblastoma cell cul- by a neuroradiologist blinded for treatment group allocation. tures (glioma-initiating cell cultures, GICs) S24 and T1 were Mice were sacrificed upon symptoms of disease in accordance established from freshly dissected glioblastoma tissue from adult with the German animal protection law. Mice with tumor patients after informed consent and cultured as described previ- growth were computationally stratified according to tumor ously (26). volumes measured in first MRI on day 14 after tumor cell implan- Cells were treated 24 hours after seeding with RG7388 tation and randomized into four treatment groups consisting of (BioVision Inc.), trametinib (Cayman Chemicals), JSH-23 vehicle control, 50 mg/kg RG7388, 1 mg/kg trametinib, or the (Cayman Chemicals), or linsitinib (OSI-906l Selleckchem), all combination of both drugs using customized R scripts. The diluted in dimethylsulfoxide (DMSO). Concentrations were vehicle consisted of (2-hydroxypropyl)-b-cyclodextrin (Sigma indicated in the respective experiments and were demonstrated Aldrich). Mice were treated daily by oral gavage for 21 days in relation to cells treated with DMSO control. For combined starting after first MRI. Changes in tumor volumes in MRI at week treatment with radiotherapy cells were irradiated with 2 and 5 in relation to baseline MRI at week 2 after tumor cell implan- 4 Gray 2 hours after treatment with RG7388 or DMSO control. tation were illustrated in the respective figure. For toxicity anal- Transient knockdown of insulin growth factor binding protein ysis body weights of mice were obtained once a week during 1 (IGFBP1) was performed with siRNA transfection (Sigma the treatment period. For analysis of on-target efficacy, mice

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brains were excised and pERK1/2 IHC was performed using anti- (1:240; R&D-systems), mouse phospho-Ika (1:1,000), rabbit pospho-MAPK antibody (1:100; Cell Signaling Technology) as Ika (1:1,000), rabbit ERK1/2 (1:1,000, all obtained from Cell described more detailed in supplementary methods. Signaling Technology). The PathScan Multiplex Western Cock- tail I (1:200; Cell Signaling Technology) contains antibodies Clonogenicity assay against phospho-ERK1/2, phospho-p90RSK, phospho-AKT Clonogenic capacity ("clonogenicity") was assessed with the (Ser473), and phospho-S6 ribosomal protein and was mainly assay appropriate for the respective cells. For the glioblastoma used to analyze phospho-ERK1/2. Equal protein loading was cell lines U87MG and A172, clonogenicity was analyzed by controlled with goat GAPDH (1:5,000; Linaris) or mouse limiting dilution assay (LDA) as described previously and a-Tubulin (1:5,000; Sigma Aldrich) staining. For trametinib analyzed using extreme limiting dilution (ELDA) software treatment, a 10-time higher concentration was used compared (26, 28). For the cell lines T98G, LN428, LN308, and LN229 with proliferation assays as cells were treated only for 24 hours clonogenicity assays were performed by seeding 500 glioma (vs. 72 hours in proliferation assays). cells in 2 mL culture medium in triplicates in six-well plates. For the GICs S24 and T1 spheroid assays were used to analyze Flow cytometry clonogenicity by seeding 150 cells in 0.2 mL of culture medium For cell cycle, analysis 100,000 cells were seeded in six-well per well in 10 wells per treatment in 96-well plates. Cells were plates and treated after 24 hours with the indicated concen- treated 24 hours after seeding as indicated in respective experi- trations of RG7388 or DMSO control. After 72 hours cells ments and number of clones per well were counted in relation were incubated in 70% ethanol, stained with 40 mg/mL to DMSO control treatment after 2 weeks (clonogenicitiy propidium iodide enriched with 20 mg/mL RNAse and assays) or 3 weeks (spheroid assays). analyzed in a BD-FACS Canto II flow cytometer. Final data were processed with FloJo flow cytometry analysis software 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Treestar). For analysis of apoptosis sub-G phase was mea- bromide (MTT) assay 1 sured in cell-cycle analysis. For MTT assay cells were seeded as described in previous section in 96-well microplates. At indicated time points MTT was added in Microarray and gene set enrichment analysis a final concentration of 5 mg/mL and absorption was analyzed at Microarray analysis was performed by the Genomics and 595 nm with a microplate reader. Proteomics Core Facility of the German Cancer Research Center 3H-Thymidine incorporation assays (Heidelberg, Germany) using Illumina HumanHT-12v4 Cells were seeded at 2,000 cells per well in 0.2 mL of culture Expression Bead Chips which analyze the expression levels of medium and treated as indicated in the respective experiments 31,000 annotated genes. Three independent samples of 24 hours after seeding. Forty-eight hours after treatment, cells RG7388 resistant and related DMSO control treated U87MG were pulsed with 3H-methylthymidine (0.5lCi; Amersham cells were analyzed for comparison. Data analysis was Radiochemical Centre, Buckinghamshire, UK) for another performed with Ingenuity Pathway Analysis (IPA; Ingenuity 24 hours and radionuclide uptake was measured by scintilla- Systems Inc.) and gene set enrichment analysis (GSEA; ref. 32). tion counting. The GSEA software tool was downloaded from the homepage of the Broad institute (http://software.broadinstitute.org/gsea). Matrigel invasion assay Fifty hallmark gene sets as well as the C2 and C5 gene sets were Matrigel invasion assay for measuring glioma cell invasion used for exploratory testing of pathway enrichments. The was performed as described previously (29, 30). For trametinib number of permutations was set to 1,000. treatment, a 10-time higher concentration was used compared with proliferation assays as cells were treated only for 24 hours Statistical analysis (vs. 72 hours in proliferation assays). For experiments with Statistical significance was assessed by Student t test (Excel; transient knockdown of IGFBP1, cells were seeded at 100,000 Microsoft) or one-sample t test as appropriate (Graph Pad cells per well in six-well plates, transfected with siRNA and added Software). P values of P < 0.05 were considered significant. to the Boyden chambers 48 hours after transfection. Figures represented summaries of at least three independent experiments in proportion to the related control, if not other- Quantitative real-time PCR wise specified.Immunoblot,qRT-PCR,andflow cytometry data RNA extraction, cDNA synthesis, and qRT-PCR were performed were shown as one representative out of three independent as previously described (31). Primer sequences are listed in experiments. Quantification of immunoblot data was perform- Supplementary Table S1. ed with ImageJ after exclusion of overexposed blots. Synergistic P53 sequencing effects were analyzed based on at least three independent For p53 sequencing, RNA and cDNA of U87MG cells that were experiments. Observed inhibition of combination therapy and (i) RG7388 resistant, (ii) DMSO control treated, and (iii) expected inhibition under independence of the individual wild-type were isolated as previous described (31). Fragments of therapies were calculated using Bliss' independence method exons 2 to 9 of the p53 gene were amplified using PCR technique (33). Synergistic effect of the combination therapy was tested and sequenced with Sanger sequencing. Primer sequences are in a linear regression model based on log-transformed inhibi- listed in Supplementary Table S1. tion measurements with main therapy effects, an interaction term between both therapies and the experiment effect to Immunoblot analysis account for paired measurements as predictors. An over- Whole cell lysates were prepared as described previously additive significant interaction was interpreted as synergism. (31). The following antibodies were used: goat IGFBP1 P values < 0.05 were considered significant.

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Results tance was maintained in RG7388 resistant cells after wit- hdrawal of permanent RG7388 exposure for three weeks (Sup- Effects of RG7388 treatment in glioblastoma cells plementary Fig. S3). Short-term treatment with 100 nmol/L RG7388 inhibited The RG7388 resistant subline showed a more aggressive clonogenicity in p53 wild-type glioblastoma cell lines (U87MG phenotype: proliferation was 1.5-times and clonogenicity and A172, Fig. 1A and Supplementary Fig. S1A) and p53 2-times increased in RG7388 resistant compared to control wild-type glioma-initiating cell cultures (GICs; Supplementary cells (Fig. 2B). Furthermore, resistant cells were 3-times more T1, Supplementary Fig. S24, Fig. 1A), whereas p53 mutant invasive (Fig. 2B). Short-term RG7388 treatment over 72 hours (LN18, LN428, U318, U373; Supplementary Fig. S1A) and led to an increase of cells in G1 cell cycle phase in long-term p53-deficient cell lines (LN308) were primary resistant against DMSO control treated cells, whereas the RG7388 resistant RG7388 treatment in a concentration of 100 nmol/L cells showed a highly increased amount of cells in G phase (Supplementary Fig. S1A). In p53 wild-type glioblastoma cell 2 after three months of RG7388 treatment (Fig. 2C). Interest- lines (U87MG, A172) and GICs (T1, S24), RG7388 led to a ingly, resistance was not limited to RG7388 treatment, but significant and dose-dependent reduction of clonogenicity and radiotherapy was also less effective with an inhibition of proliferation [Fig. 1A; Supplementary Fig. S2, left panel ("no proliferation by 30% at 2 Gy and 56% at 4 Gy in RG7388 RT") of each graph]. In U87MG cells, RG7388 induced protein resistant cells in comparison to an inhibition by 45% at 2 Gy levels of p53 target genes, such as p21 and MDM2 and 80% at 4 Gy in DMSO treated control cells (Fig. 2D). (Supplementary Fig. S1B), apoptosis (Supplementary To identify potential resistance mechanisms, microarray Fig. S1C), and a G arrest (Supplementary Fig. S1D). 1 analysis was performed comparing RG7388-resistant U87MG cells with the respective control cells. Microarray analysis Synergism of RG7388 treatment and radiotherapy revealed an activation of extracellular signal-regulated kinases fi As radiotherapy is the standard of care for rst-line treatment 1/2 (ERK1/2) and NF-kB pathway in RG7388-resistant cells, of patients with glioblastoma, we analyzed potential synergistic which was confirmed by immunoblot analysis (Fig. 2E). Fur- effects of RG7388 therapy and radiotherapy at clinically relevant thermore, among the 20 most regulated genes in microarray radiation doses. Combined treatment of RG7388 in low nano- analysis (Table S2) IGFBP1 (upregulated by 8-fold in RG7388 – molar concentrations (10 100 nmol/L) and radiotherapy at resistant cells) was the most promising candidate due to the 2 and 4 Gy showed synergistic effects on the inhibition of strongest reliability of confirmation by qRT-PCR (Supplemen- clonogenicity (Fig. 1A, middle and right panel, respectively). tary Fig. S6A) and immunoblot analysis (Fig 2E) as well as Calculated expected inhibition of combined treatment based based on literature research demonstrating a cross-link with the on impacts of respective monotherapies and observed inhibi- ERK1/2 pathway (34, 35). In addition, microarray analysis tion of combined treatment as well as P values of over-additive showed an activation of the p53 pathway in RG7388-resistant fi interaction are demonstrated in respective gures to illustrate cells, which was also seen in GSEA (Fig. 2F). Of note, p53 synergism. As an example, 50 nmol/L RG7388 treatment alone sequencing in DMSO control treated and RG7388 resistant reduced clonogenicity in S24 cells by 33%, 2 Gy by 32%, and cells confirmed the maintenance of p53 wild-type status in 4 Gy by 66%. The combination of 50 nmol/L RG7388 and 2 Gy these cells. inhibited clonogenicity by 86% and in combination with 4 Gy by 94%. Calculated expected inhibition based on effects Restoring RG7388 sensitivity in RG7388-resistant cells of respective monotherapies were 68% for treatment with To identify rational treatment strategies to overcome RG7388 50 nmol/L RG7388 combined with 2 Gy radiotherapy and resistance, the activated NF-kB and ERK pathways and the 83% for combination with 4 Gy. Therefore, these data repre- enhanced IGFBP1 expression were inhibited. NF-kB inhibitor sented strong synergistic effects of RG7388 treatment and radio- JSH-23 treatment at 10 mmol/L for 72 hours (Fig. 3A), but also < therapy (both P 0.001, respectively; Fig. 1A; Supplementary at up to 30 mmol/L, did not relevantly alter proliferation as Fig. S24 cells). These synergistic effects were substantiated monotherapy. Although combined JSH-23 and RG7388 treat- when examining the reduction of proliferation in the p53 ment revealed significant additive effects on inhibition of prolif- wild-type GICs and glioblastoma cell lines (Supplementary eration, over-additive effects were not observed as sign for over- Fig. S2). Cell viability measurements in freshly isolated murine coming RG7388 resistance. Likewise, combining RG7388 and astrocytes and neurons did not reveal relevant toxicity of radiotherapy, which was tested based on the synergistic impact RG7388 monotherapy on murine normal brain cells in-vitro. seen in nonresistant cells, did not overcome resistance. Radio- Although irradiation led to a small, but not relevant, reduction therapy on its own was slightly anti-proliferative in RG7388- of cell viability, combined treatment with RG7388 did not resistant cells, but the addition of RG7388 had no additional further increase toxicity in these cells (Fig. 1B). effect (Fig. 3B). Inhibition of IGFBP1 by transient knockdown via siRNA Resistance against RG7388 treatment revealed synergistic effects in combination with short-term Resistance against targeted therapies is a main issue in the treatment with 100 nmol/L RG7388 in RG7388-resistant development of clinically effective treatments. To analyze pos- U87MG cells. Although transient IGFBP1 knockdown alone sible resistance mechanisms of chronic RG7388 exposure, did not relevantly change proliferation of RG7388-resistant RG7388 resistant cells were generated by treating U87MG cells cells, a combined treatment inhibited proliferation by with increasing doses of RG7388 up to 10 mmol/L over a period 50% (Fig. 3C). In contrast, transient knockdown of IGFBP1 of three months. Resistance of these cells against RG7388 was alone significantly ameliorated the increased invasion in confirmed by reduced impact on clonogenicity after short-term RG7388-resistant cells to the level seen in DMSO control treatment with RG7388 (Fig. 2A). In addition, RG7388 resis- cells (Fig. 3D).

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Figure 1. Synergistic effects of combined RG7388 treatment and radiotherapy on inhibition of clonogenicity. A, RG7388 dose-dependently reduced clonogenicity in p53 wild-type glioma initiating cell cultures (GICs; S24, T1) and glioblastoma cell lines (U87MG, A172) and showed significant synergistic effects in combination with radiotherapy. B, RG7388 alone and in combination with radiotherapy revealed no relevant toxicity on freshly isolated murine astrocytes and neurons. Figures represent mean values of at least three independent experiments. demonstrates levels of significance calculated between RG7388 treatment and related DMSO control, indicates levels of significance of synergistic effects of combined RG7388 treatment and radiotherapy. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 2. Long-term RG7388 treatment resulted in resistance against RG7388 in U87MG cells ("RG7388 resistant cells"). A, Short-term RG7388 treatment relevantly reduced stem cell frequency in DMSO-treated control cells (DMSO control), but not in RG7388-resistant cells. B, RG7388 resistant cells were signiﬁcantly more proliferative, more clonogenic, and more invasive than DMSO treated control cells. C, Although short-term treatment with RG7388

for 72 hours led to an increase of cells in G1 cell-cycle phase, long-term RG7388 treatment over 3 months increased amount of cells in G2 phase. D, RG7388- resistant cells were also more resistant against radiotherapy. E, Immunoblot analysis showed a relevant activation of ERK1/2 and NF-kB pathway and an upregulation of IGFBP1 in RG7388-resistant U87MG cells. F, GSEA revealed a higher activation of p53 pathway in RG7388 resistant cells compared with DMSO control cells. Figures represent mean values of at least three independent experiments. Only immunoblot and cell-cycle data represent one experiment out of three independent experiments. indicates signiﬁcance level tested in comparison to the related control. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 3. Restoring RG7388 sensitivity in RG7388-resistant U87MG cells. AþB, Neither inhibition of NF-kBpathwayby10mmol/L JSH-23 (A) nor radiotherapy (B) in combination with RG7388 treatment was able to overcome RG7388 resistance. C, Transient knockdown of IGFPB1 did not relevantly alter proliferation, but revealed significant synergistic effects in combination with short-term RG7388 treatment at RG7388 resistance. D, Transient IGFBP1 knockdown reduced invasiveness of RG7388-resistant cells to a comparable level with DMSO control cells. E, Inhibition of the ERK1/2 pathway by trametinib showed significant synergistic effects on reduction of proliferationincombinationwithRG7388treatment.F, Trametinib treatment reduced the invasive phenotype of RG7388 resistant cells. Because of shorter treatment compared to proliferation assays (24 hours vs. 72 hours) higher concentrations of trametinib were used in migration assay. Figures represent mean values of at least three independent experiments. indicates the level of significance compared to related control; demonstrates level of significance of synergistic effects. , P < 0.05; , P < 0.01; , P < 0.001; n.s., not significant; tram., Trametinib.

Inhibition of the ERK1/2 signaling pathway by the MEK inhib- therapy with inhibition of proliferation by 51% at 1 nmol/L itor trametinib as monotherapy had no relevant impact on trametinib and 61% at 2.5 nmol/L trametinib in combination proliferation of RG7388-resistant U87MG cells in the nanomolar with 100 nmol/L RG7388 (Fig. 3E). Furthermore, trametinib concentrations tested. However, in contrast to NF-kB inhibition at 10 nmol/L reduced in particular the proinvasive phenotype and radiotherapy, trametinib restored sensitivity toward RG7388 of RG7388-resistant cells leading nearly to normalization of

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Figure 4. Combined RG7388 and trametinib treatment significantly inhibited tumor growth of RG7388- resistant U87MG cells in vivo, whereas RG7388 monotherapy at 50 mg/kg and trametinib monotherapy at 1 mg/kg had no relevant impact on tumor growth of RG7388-resistant cells in vivo,the combined treatment significantly inhibited tumor growth in these tumors. MRIs were performed at week 2 ("baseline MRI") and week 5 after implantation of tumor cells. Changes in tumor volumes in the MRI at week 5 in relation to respective tumor volumes in baseline MRI are demonstrated in the top part of the figure. Significant differences in tumor growth are indicated with P < 0.05. Representative MRI images of the different treatment groups are illustrated in the lower part of the figure. tram., trametinib.

invasiveness when compared with DMSO control treated cells available safety data from clinical trials with respective mono- (Fig. 3F). Moreover, short-term treatment (for 72 hours) of both therapies (13, 36, 37). trametinib and RG7388 also showed synergistic effects in A172 and U87MG wild-type cells (Supplementary Fig. S4). Resistance is mediated via IGFBP1–ERK1/2 signaling cascade Animal experiments performed with the engineered When further investigating the signaling pathways affected RG7388-resistant U87MG cells orthotopically implanted in by the effective treatments described, immunoblot analysis immunodeficient mice confirmed the relevant synergistic revealed that transient knockdown of IGFBP1 reduced not effects of combined trametinib and RG7388 treatment at only IGFBP1 expression but also the activation of ERK1/2 RG7388 resistance (Fig. 4), whereas respective monotherapy signaling pathway in DMSO control treated and RG7388- did not significantly reduce growth of these tumors. Comparing resistant cells (Fig. 5A). Vice versa, short-term treatment with changes of tumor volumes in MRI at week 5 after tumor cell trametinib reduced IGFBP1 expression in RG7388-resistant implantation (at week 3 after treatment start) to respective cells (Fig. 5B). IGFBP1 binds the IGF receptor leading to an tumor volumes in baseline MRI, tumor growth was inhibited by activation of the ERK1/2 signaling pathway (38). The latter was 9% with RG7388 monotherapy and 11% with trametinib confirmed for the U87MG RG7388 resistant cells. The IGFR monotherapy compared with vehicle control treatment (both inhibitor linsitinib reduced the activation of ERK1/2 pathway not significant). In contrast, combined RG7388 and trametinib particularly in the U87MG RG7388 resistant cells (Fig. 5C). treatment reduced tumor growth by 67% compared with vehi- To further analyze the interaction of IGFBP1 and ERK1/2 cle control (P ¼ 0.012). Moreover, combined treatment signaling on a molecular level, transcription factors with pre- revealed a significant higher reduction of tumor growth com- dicted binding sites at the IGFBP1 promotor were searched in pared with RG7388 monotherapy (P ¼ 0.01) and trametinib the transcription factor target gene data base (39) and screened monotherapy (P ¼ 0.017). IHC analysis showed a reduction of for an upregulation in RG7388-resistant cells based on micro- phosphor-ERK1/2 after trametinib treatment as demonstration array data. A significant upregulation in RG7388 resistant of on-target efficacy (Supplementary Fig. S5B). Relevant toxi- compared with DMSO control cells was validated for the cities were not observed with monotherapies nor combined transcription factors ZIC2 and NR2F1 by qRT-PCR. In addition, treatment based on changes in animal weights (Supplementary expressionofZIC2andNR2F1wassignificantly reduced by Fig. S5A). The good tolerability is further substantiated by trametinib treatment (Fig. 5D) as well as by knockdown of

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Figure 5. Signaling pathways of ERK1/2 and IGFBP1 inhibitory strategies. A, Activation of ERK1/2 pathway was inhibited by transient knockdown of IGFBP1 in U87MG DMSO control and RG7388 resistant. B, Vice versa, ERK1/2 signaling pathway and IGFBP1 expression was reduced by treatment with trametinib in U87MG DMSO- treated control and RG7388-resistant cells. C, Inhibition of IGF receptor by the IGFR inhibitor linsitinib reduced ERK1/2 signaling activation particularly in U87MG RG7388-resistant cells. D and E, MRNA expression of the transcription factors ZIC2 and NR2F1 were significantly upregulated in U87MG RG7388-resistant cells and significantly reduced by either trametinib treatment (D) or IGFBP1 knockdown (F). G, Short-term RG7388 treatment led to a significant upregulation of IGFBP1 mRNA expression in U87MG cells, but did not result in a relevant upregulation of IGFBP1 protein expression or further activation of ERK1/2 signaling. H, Schematic description of molecular mechanisms of RG7388 resistance: Short-term RG7388 treatment increased IGFBP1 mRNA expression probably via the p53 binding site at the IGFBP1 promotor, but did not further activate the ERK1/2 signaling pathway. At RG7388 resistance, the IGFBP1 mRNA and protein expression was highly upregulated leading to an activation of ERK1/2 signaling and upregulation of ZIC2 and NR2F1 mRNA expression. As the latter represent candidates for transcription factors with predicted binding sites at the IGFBP1 promotor the upregulation of ZIC2 and NR2F1 expression might further increase the IGFBP1 expression. This proposed self-activating bypass resistance signaling pathway was inhibited by trametinib treatment or IGFBP1 knockdown resulting in reduced proliferation and invasion of RG7388-resistant cells. Immunoblot results are quantified and demonstrated as proportions to related controls ("si-control" for A, "DMSO" for B, C, and G). Figures demonstrate one experiment out of three independent experiments. tram., trametinib; linsi., linsitinib.

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IGFBP1 (Fig. 5E). Knockdown of IGFBP1 and efficacy of tra- Regarding acquired resistance mechanisms, previous studies metinib treatment was verified by a reduction of IGFBP1 of different MDM2 inhibitors in other tumor entities strongly expression in RG7388-resistant cells by qRT-PCR (Supplemen- indicate that resistant cells harbor p53 mutations, which are tary Fig S7). Furthermore, TP53 was also found to have a acquired during long-term treatment with MDM2 inhibitors predicted binding site at the IGFBP1 promotor. Short-term (21–24). In contrast to these data, resistance of RG7388 long- RG7388 treatment increased IGFBP1 mRNA expression, but term treated U87MG glioblastoma cells was not mediated via did not result in a relevant increase of IGFBP1 protein expres- acquisition of p53 mutations. Microarray and gene enrichment sion and further activation of ERK1/2 signaling (Fig. 5F). In analysis demonstrated an activation of p53 pathway in contrast, the previous shown stronger activation of the p53 RG7388-resistant compared with DMSO control treated cells. pathway at RG7388 resistance led to a significant upregulation Furthermore, microarray analysis revealed an upregulation of of IGFBP1 protein expression (Fig. 2E), resulting in an activa- IGFBP1 expression as well as an activation of ERK1/2 and tion of ERK1/2 signaling and upregulation of the transcription NF-kB pathway in RG7388-resistant cells. factors ZIC2 and NR2F1, which then might further enhance Inhibition of NF-kB pathway demonstrated additive effects the IGFBP1 expression though the binding sites at the IGFBP1 in combination with RG7388 but did not restore sensitivity protomor. This self-activating pathway could be inhibited by towards RG7388 treatment. In contrast, inhibition of ERK1/2 trametinib treatment or IGFBP1 knockdown, which both pathway by the MEK inhibitor trametinib dose-dependently resulted in reduced IGFBP1 expression, reduced activation of overcame RG7388 resistance and reduced the highly invasive ERK1/2 and reduced expression of the transcription factors phenotype of resistant cells. In vivo experiments further sub- ZIC2 and NR2F1 (Fig. 5G). stantiated the relevant benefit of combined RG7388 and tra- IGFBP1 is strongly upregulated in RG7388-resistant U87MG metinib treatment as salvage therapy at RG7388 resistance cells, but basal expression levels in glioblastoma cell lines and demonstrated by a significant higher reduction of tumor GICs are relatively low (Supplementary Fig. S6B). Therefore, a growth with combined treatment compared with respective stable IGFBP1 OE was induced in the p53 wild-type glioblas- monotherapies and vehicle control treated mice. toma cell lines U87MG and A172 to re-evaluate the results in In clinical practice, treatment is often stopped after the emer- othercells.IGFBP1OEwasconfirmed by immunoblot analysis gence of resistance and replaced by another salvage therapy. (Fig. 6A) and resulted in a higher resistance against short-term However, the significant synergistic effects of combined treat- RG7388 treatment compared with vector control transfected ment strongly suggest that RG7388 treatment may be continued cells (Fig. 6B), which could be restored by transient knockdown and the combination with trametinib should be further of IGFBP1 (Fig. 6C). In accordance with the data on long-term explored. Of note, also short-term trametinib and RG7388 treatment with RG7388 in U87MG cells ("RG7388 resistant treatment was synergistic in A172 and U87MG wild-type cells. cells"), trametinib treatment also showed synergistic effects in Hata and colleagues demonstrated synergistic effects of MDM2 combination with 100 nmol/L RG7388 in IGFBP1 overexpres- and MEK inhibition in KRAS mutant non–small cell lung cancer sing cells (Fig. 6D). and colorectal cancer as first-line therapy (24). Therefore, it remains to be determined if combined first-line treatment may delay RG7388 resistance or results in heterogeneous resistance Discussion to one or both of the targeted compounds (24). Primary and acquired resistances are a big challenge and Inhibition of IGFBP1 expression by transient knockdown limitation for the effective clinical use of targeted therapies. normalized increased invasiveness in RG7388-resistant cells Although primary resistance mechanisms are widely studied for and reduced proliferation in combination with RG7388 in a MDM2 inhibitors in different tumor entities (7, 17, 18, 40), synergistic manner. Studies in hepatocytes and colon carcino- acquired resistance mechanisms are still not fully understood ma cells suggested a crosslink between IGFBP1 and ERK1/2 and were not in the focus in neuro-oncology so far. signaling (34, 35), which was confirmed in our cells. Trame- Regarding primary resistance, the in vitro data reconfirm that tinib treatment reduced IGFBP1 expression and vice versa, p53 wild-type status is a prerequisite for sensitivity to RG7388 transient IGFBP1 knockdown inhibited ERK1/2 pathway acti- treatment in glioblastoma. Conclusively, these data further sup- vation. On a molecular level, mRNA expressions of ZIC2 and port the use of p53 mutation status as a biomarker for primary NR2F1 as candidates for transcription factors with binding sites response to treatment with MDM2 inhibitors. Furthermore, at the IGFBP1 promotor were upregulated in RG7388 resistant RG7388 acts synergistically with radiotherapy in p53 wild-type cells and inhibited by trametinibtreatmentandIGFBP1knock- glioblastoma cell lines and primary GICs. Combined treatment down. These data further confirm the cross-link between did not demonstrate relevant effects in freshly isolated murine IGFBP1 and ERK1/2 signaling and demonstrate a self-activating neurons and astrocytes, which were used to analyze possible off- pathway as a bypass resistance signaling. Short-term treatment target toxicity. The lack of impact compared to tumor cells might with RG7388 led to an upregulation of IGFBP1 mRNA expres- be due to the low proliferation rate and low clonogenicity of sion probably via the TP53 binding site at the IGFBP1 promo- neurons and astrocytes. There is no reason to expect toxicity in tor, but did not result in a relevant upregulation of IGFBP1 resting human cells as well. In conclusion, these preclinical data protein expression and further activation of ERK1/2 signaling. warrant further clinical development to study the combination of Upon RG7388 resistance, the TP53 pathway was stronger MDM2 inhibition and radiotherapy as first-line therapy in glio- activated compared with control cells, which resulted in a blastoma patients harboring a p53 wild-type status (41). In higher induction of IGFBP1 mRNA and protein expression and addition, the data validate and relevantly extend the previous further activation of ERK1/2 pathway probably via the IGF stated enhancement of radiosensitivity with nulin-3a in U87MG receptor as inhibition of IGFR reduced ERK1/2 signaling acti- glioblastoma cells (19). vation particularly in the RG7388-resistant cells. Activation of

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Figure 6. RG7388 resistance is mediated via activation of the IGFBP1–ERK1/2 signaling pathway. A and B, Exogenous IGFBP1 OE in A172 and U87MG wild-type cells was confirmed by immunoblot analysis (A) and resulted in resistance against short-term RG7388 treatment in relation to vector control transfected cells (B). C and D, Short-term RG7388 treatment combined with transient knockdown of IGFBP1 (C) or in combination with short-term trametinib treatment (D)showedsignificant synergistic effects in IGFBP1 overexpressing A172 and U87MG cells. Figures represent mean values of at least three independent experiments. demonstrates levels of significance calculated between RG7388 treatment and related DMSO control, indicates levels of significanceofsynergisticeffectsofcombinedtreatment., P < 0.05; , P < 0.01; , P < 0.001. tram., trametinib.

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the IGFBP1–ERK1/2 signaling further increased the expression tinued and combined with trametinib rather than discontinued of the transcription factors ZIC2 and NR2F1, which then might after resistance against RG7388 has occurred. Furthermore, in increase the IGFBP1 expression via the binding sites at IGFBP1. view of the relevant synergistic effects, the data further support the This self-activating pathway could be inhibited by trametinib combination of radiotherapy and RG7388 treatment in first-line treatment as well as by IGFBP1 knockdown resulting in a therapy especially in a situation, when temozolomide as the reduction of proliferation and invasion in RG7388-resistant standard alkylating drug is of no value because of O6- cells. Furthermore, the cross-link to the p53 pathway could methylguanine DNA-methyltransferase promoter methylation, explain the synergistic effects of RG7388 and trametinib treat- whereas re-irradiation seems not to be an effective salvage therapy ment or IGFBP1 knockdown. After inhibition of the IGFBP1– after development of RG7388 resistance. RG7388 belongs to the ERK1/2 pathway by the treatments described additional targeted therapies evaluated in the ongoing NCT Neuro Master RG7388 treatment might again be able to mainly activate the Match (N2M2) phase I/IIa clinical trial (NCT03158389), which p53 pathway resulting in an effective reduction of proliferation intends to personalize treatment options based on molecular and invasion at RG7388 resistance. profiling for glioblastoma patients with an unmethylated MGMT IGFBP1 expression is generally low in glioblastoma cells but promotor (41). This is an ideal opportunity to investigate whether highly upregulated in RG7388-resistant cells. Interestingly, exog- the described mechanism of resistance can be substantiated enous OE of IGFBP1 in A172 and U87MG resulted in develop- and the proposed salvage therapies holds true in relapsing ment of a more resistant phenotype towards RG7388. In accor- glioblastoma after RG7388 treatment. dance with the previous described data in RG7388 resistant U87MG cells, combined treatment with either trametinib or Disclosure of Potential Conflicts of Interest transient knockdown of IGFBP1 restored sensitivity toward No potential conflicts of interest were disclosed. RG7388 therapy in IGFBP1 overexpressing cells. Therefore, these data further confirm the activation of the ERK1/2–IGFBP1 sig- Authors' Contributions naling cascade as a key mechanism for resistance against RG7388. Conception and design: A. Berberich, W. Wick In contrast to the synergistic effects of radiotherapy and Developmentofmethodology:A. Berberich, S. Ciprut, D. Lemke, RG7388 treatment at first-line therapy, radiotherapy was not A. von Deimling effective in RG7388-resistant cells making re-irradiation as salvage Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Berberich, T. Kessler, S. Pusch, F. Sahm, therapy less attractive. L.-M.Schmitt,N.Hucke,P.Ruebmann,M.Fischer,M.O.Breckwoldt, Long-term treatment often results in selection pressure for more A. von Deimling, M. Bendszus, W. Wick aggressive tumor cells causing problems for effective salvage Analysis and interpretation of data (e.g., statistical analysis, biostatistics, therapies. In this study, RG7388 resistant cells showed a more computational analysis): A. Berberich, T. Kessler, C.M. Thome, S. Pusch, aggressive phenotype than related control cells with a higher T. Hielscher, F. Sahm, A. von Deimling, M. Bendszus, W. Wick clonogenicity, proliferation and invasiveness demonstrating the Writing, review, and/or revision of the manuscript: A. Berberich, T. Kessler, S. Pusch, M.O. Breckwoldt, A. von Deimling, M. Bendszus, M. Platten, W. Wick importance for rational, effective and for clinical use suitable Administrative, technical, or material support (i.e., reporting or organiz- salvage therapies. ing data, constructing databases): C.M. Thome, I. Oezen, M. Fischer, In summary, this study presents a mechanism of acquired D. Lemke, A. von Deimling, M. Bendszus, W. Wick resistance against MDM2 inhibitors in glioblastoma, suggesting Study supervision: W. Wick rationales for salvage therapies which should be evaluated in clinical practice. In p53 wild-type glioblastoma cells, RG7388 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked resistance was mediated via activation of the ERK1/2–IGFBP1 advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate signaling cascade, which was effectively targetable by the clinical this fact. approved MEK inhibitor trametinib. The data demonstrated a relevant benefit of combined trametinib and RG7388 treatment at Received May 20, 2018; revised August 7, 2018; accepted September 27, 2018; RG7388 resistance implying that RG788 therapy should be con- published first October 1, 2018.

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Targeting Resistance against the MDM2 Inhibitor RG7388 in Glioblastoma Cells by the MEK Inhibitor Trametinib

Anne Berberich, Tobias Kessler, Carina M. Thomé, et al.

Clin Cancer Res Published OnlineFirst October 1, 2018.

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