targeting resistance against the mdm2 inhibitor rg7388 in ......sep 29, 2018 · dysregulation of...

1

Targeting resistance against the MDM2 inhibitor RG7388 in glio-

blastoma cells by the MEK inhibitor trametinib

Anne Berberich1,2, Tobias Kessler1,2, Carina M. Thomé1, Stefan Pusch3,4, Thomas Hielscher5,

Felix Sahm4, Iris Oezen6, Lara-Marie Schmitt1,2, Sara Ciprut1, Nanina Hucke1,2, Petra Rueb-

mann1, Manuel Fischer8, Dieter Lemke1,2, Michael O. Breckwoldt6,8, Andreas von Deimling3,4,

Martin Bendszus8, Michael Platten2,6,7, Wolfgang Wick1,2

1Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German

Cancer Research Center (DKFZ)

2Department of Neurology, Heidelberg University Hospital

3Clinical Cooperation Unit Neuropathology, DKTK, DKFZ

4Department of Neuropathology, Heidelberg University Hospital

5Division of Biostatistics, DKFZ

6Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, DKTK, DKFZ, all

Heidelberg, Germany

7Department of Neurology, Medical Faculty Mannheim, Heidelberg University, Mannheim,

Germany

8 Department of Neuroradiology, Heidelberg University Hospital

Correspondence to:

Wolfgang Wick, MD

Neurology Clinic & Neurooncology Program at the National Center for Tumor Diseases

Im Neuenheimer Feld 400

D-69120 Heidelberg, Germany

Phone: +496221567075

Fax: +496221567554

E-mail: [email protected]

Running title:

Acquired resistance against RG7388 in glioblastoma

Key words:

Glioblastoma, MDM2, RG7388, resistance, radiotherapy

Research. on March 18, 2021. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 1, 2018; DOI: 10.1158/1078-0432.CCR-18-1580

http://clincancerres.aacrjournals.org/

2

Conflict of interest statement: None of the authors has a conflict of interest in the topic of

this manuscript or related topics.

Word count (abstract): 245

Total words of main text (without references): 4996

References: 41

Number of figures: 6

Statement of translational relevance

The present experiments provide a good rational for a novel systemic treatment, that is radio-

therapy, MDM2 and ERK1/2 inhibition specifically in the so far underserved group of glioblas-

toma patients with a lack of MGMT promoter hypermethylation. As is, the combination will be

forwarded to the clinic in a multicenter phase I/II clinical trial with molecular preselection of

suitable patients.




3

Abstract

Purpose: Resistance is an obstacle of glioma therapy. Despite targeted interventions, tu-

mors harbor primary resistance or become resistant over short course of treatment. This

study examined the mouse double minute 2 (MDM2) inhibitor RG7388 together with radio-

therapy and analyzed strategies to overcome acquired MDM2 inhibitor resistance in glioblas-

toma.

Experimental Design: Effects of RG7388 and radiotherapy were analyzed in p53 wild-type

glioblastoma cell lines and glioma-initiating cells. RG7388 resistant cells were generated by

increasing RG7388 doses over three months. Regulated pathways were investigated by mi-

croarray, qRT-PCR and immunoblot analysis and specifically inhibited to evaluate rational

salvage therapies at RG7388 resistance. Effects of RG7388 and trametinib treatment were

challenged in an orthotopical mouse model with RG7388 resistant U87MG glioblastoma

cells.

Results: MDM2 inhibition required functional p53 and showed synergistic activity with radio-

therapy in first-line treatment. Long-term exposure to RG7388 induced resistance by activa-

tion of the extracellular signal-regulated kinases 1/2 (ERK1/2) - insulin growth factor binding

protein 1 (IGFBP1) signaling cascade, which was specifically overcome by ERK1/2 pathway

inhibition with trametinib and knockdown of IGFBP1. Combining trametinib with continued

RG7388 treatment enhanced anti-tumor effects at RG7388 resistance in vitro and in vivo.

Conclusions: These data provide a rationale for combining RG7388 and radiotherapy as

first-line therapy with a specific relevance for tumors insensitive to alkylating standard chem-

otherapy and for the addition of trametinib to continued RG7388 treatment as salvage thera-

py after acquired resistance against RG7388 for clinical practice.




4

Introduction

Dysregulation of the p53 pathway is found in 85.3% of primary glioblastoma and caused by

p53 mutation or homozygous deletion (27.9%), deletion of cyclin-dependent kinase inhibitor

2A (CDKN2A) (57.8%) or amplification of mouse double minute homologs 1/2/4 (MDM1/2/4)

(15%) (1). Overexpression of MDM2, the key negative regulator of p53, impairs p53 wild-type

function and deregulates the MDM2-p53 feedback loop, which results in an accelerated tu-

mor growth in a variety of human tumors, including sarcoma, leukemia, breast cancer, mela-

noma and glioblastoma (2-7). Targeting MDM2 evolved as a promising treatment approach

to reactivate the p53 pathway (8) leading to cell cycle arrest, increased apoptosis and de-

creased tumor growth in human tumor xenografts in nude mice (9, 10). RG7388, also known

as idasanutlin, is a second generation MDM2 inhibitor of the nutlin family with superior po-

tency and selectivity compared to its predecessor RG7112 (11). RG7388 binds selectively

and with a high affinity to the p53 binding site on the surface of the MDM2 molecule by mim-

icking the three key binding amino acids (9, 10, 12) and thereby inhibiting the MDM2-p53

interaction. There are first signs of efficacy for MDM2 inhibitors, including RG7388, in studies

for patients with leukemia and sarcoma (4, 5, 13). Clinical trials using MDM2 inhibitors for

patients with glioblastoma have not yet been conducted.

Primary and acquired resistance as well as optimal patient selection are the biggest chal-

lenges for the clinical use of targeted therapies. For glioblastoma, O6-methylguanine DNA

methyltransferase (MGMT) promoter methylation status may be regarded as the only predic-

tive biomarker aiding the decision for the use of alkylating chemotherapy. However, today’s

guidelines still advocate the use of temozolomide regardless of MGMT status and alterna-

tives for patients not likely benefitting from temozolomide are yet to be developed (14-16).

With regards to MDM2 inhibitors, several preclinical studies showed that MDM2 inhibitors

reduced tumor growth in p53 wild-type tumors, whereas tumors harboring p53 mutations

were primary resistant against the treatment (17-19). Furthermore, MDM2 amplification in

p53 wild-type tumors increased sensitivity to MDM2 inhibitory treatment strategies highlight-

ing MDM2 amplification and p53 wild-type status as potential biomarkers for patient selection




5

(7, 20). Acquired resistance mechanisms are still not understood, especially in glioblastoma.

Potential mechanisms leading to acquired MDM2 inhibitor resistance are p53 mutations (21-

24) as well as enhanced B-cell lymphoma-extra large (Bcl-xl) or MDM4 protein expression,

offering the opportunity to be targeted by the addition of specific inhibitors (25).

In the present study, we determined a so far unknown resistance mechanism against the

MDM2 inhibitor RG7388 in p53 wild-type glioblastoma cells resulting in the identification of a

new rational salvage therapy to potentially overcome resistance.

Material and methods

A more detailed description is given in the supplementary methods.

Cell culture

The human glioblastoma cell lines U87MG, A172, T98G, LN428, LN308 (ATCC; Manassas,

USA) and LN229 (N. de Tribolet, Lausanne, Switzerland), were kept under standard condi-

tions. The primary glioblastoma cell cultures (glioma initiating cell cultures, GICs) S24 and T1

were established from freshly dissected glioblastoma tissue from adult patients after in-

formed consent and cultured as described previously (26).

Cells were treated 24h after seeding with RG7388 (BioVision Inc., Milpitas, USA), trametinib

(Cayman Chemicals, Ann Arbor, USA), JSH-23 (Cayman Chemicals, Ann Arbor, USA) or

linsitinib (OSI-906, Selleckchem, Munich, Germany), all diluted in dimethylsulfoxide (DMSO).

Concentrations were indicated in the respective experiments and were demonstrated in rela-

tion to cells treated with DMSO control. For combined treatment with radiotherapy cells were

irradiated with 2 and 4 Gray two hours after treatment with RG7388 or DMSO control. Tran-

sient knockdown of insulin growth factor binding protein 1 (IGFBP1) was performed with

siRNA transfection (Sigma Aldrich) using Lipofectamine (Invitrogen, Carlsbad, USA) accord-

ing to the manufactures protocol. For stable overexpression with IGFBP1, Gateway® com-

patible IGFBP1 cDNA [NM_000596] and the vector pMXS-GW-IRES-PuroR were obtained




6

from the German Cancer Research Center clone repository. Correct sequence of cDNA was

validated by Sanger sequencing (GATC, Köln, Germany).

RG7388 resistant cells were generated by twice weekly treatment of U87MG cells with in-

creasing doses of 10 nM up to 10 µM RG7388 (“RG7388 resistant cells”) or related DMSO

amounts (“DMSO control treated cells”) over a period of three months.

Murine cerebellum neurons were freshly isolated from P6 neonatal mice. Cerebelli were di-

gested with typsine / DNAse solution as described more detailed in supplementary methods.

Cells were seeded in poly-L-lysine (PLL) coated 96-well microplates at 50,000 cells per well

in 200 µl culture media. Treatment was added 24h after seeding as indicated in respective

figures and MTT assay was performed 6 days after treatment.

Murine astrocytes were freshly isolated from P1 or P2 neonatal mice as described previously

(27). Cells were seeded in PLL coated 96-well microplates at 15,000 cells per well in 200 µl

complete DMEM media and treated 24h later as indicated in the respective figures. After

another 8 days MTT assay was performed.

Orthotopical xenograft mouse model

All animal work was approved by the governmental authorities (animal application number:

G210-16, Regierungspräsidium Karlsruhe, Germany) and performed in accordance with the

German animal protection law. 1x105 RG7388 resistant U87MG cells were stereotactically

implanted into the right brain hemisphere of deeply anesthetized CD1 nu/nu mice (Charles

River Laboratories, Sulzfeld, Germany). Size calculations for the animal experiment were

performed using G*Power calculator version 3.1.9.2 (Universität Düsseldorf, Germany) with

setting of the following variables: α = 0.05, power(β) = 0.80, estimated standard deviation =

30% of volume. A meaningful biological difference was assumed at 50% reduction in tumor

volume and the dropout rate due to lack of tumor growth was estimated as 20%. Altogether

tumor cell inoculation was carried out in 40 animals. MRIs were performed with a 9.4-Tesla




7

horizontal-bore small animal MRI scanner (BioSpec 94/20 USR; Bruker BioSpin GmbH) with

a four-channel phased-array surface receiver coil. A T2-weighted (T2-w) rapid acquisition

with relaxation enhancement (RARE) sequence was used to assess tumor volume. Tumor

segmentation was performed in Amira (FEI, Hilsboro, USA) by a neuroradiologist blinded for

treatment group allocation. Mice were sacrificed upon symptoms of disease in accordance

with the German animal protection law. Mice with tumor growth were computationally strati-

fied according to tumor volumes measured in first MRI on day 14 after tumor cell implantation

and randomized into four treatment groups consisting of vehicle control, 50 mg/kg RG7388, 1

mg/kg trametinib or the combination of both drugs using customized R scripts. The vehicle

consisted of (2-Hydroxypropyl)-β-cyclodextrin (Sigma Aldrich, USA). Mice were treated daily

by oral gavage for 21 days starting after first MRI. Changes in tumor volumes in MRI at week

5 in relation to baseline MRI at week 2 after tumor cell implantation were illustrated in the

respective figure. For toxicity analysis body weights of mice were obtained once a week dur-

ing the treatment period. For analysis of on-target efficacy, mice brains were excised and

pERK1/2 immunohistochemistry was performed using anti-pospho-MAPK antibody (1:100,

Cell Signaling) as described more detailed in supplementary methods.

Clonogenicity assay

Clonogenic capacity (“clonogenicity”) was assessed with the assay appropriate for the re-

spective cells. For the glioblastoma cell lines U87MG and A172, clonogenicity was analyzed

by limiting dilution assay (LDA) as described previously and analyzed using extreme limiting

dilution (ELDA) software (26, 28). For the cell lines T98G, LN428, LN308 and LN229 clono-

genicity assays were performed by seeding 500 glioma cells in 2 ml culture medium in tripli-

cates in 6-well plates. For the GICs S24 and T1 spheroid assays were used to analyze

clonogenicity by seeding 150 cells in 0.2 ml of culture medium per well in 10 wells per treat-

ment in 96-well plates. Cells were treated 24h after seeding as indicated in respective exper-

iments and number of clones per well were counted in relation to DMSO control treatment

after 2 weeks (clonogenicitiy assays) or 3 weeks (spheroid assays).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay




8

For MTT assay cells were seeded as described in previous section in 96-well microplates. At

indicated time points MTT was added in a final concentration of 5 mg/ml and absorption was

analyzed at 595 nm with a microplate reader.

³H-Thymidine incorporation assays

Cells were seeded at 2,000 cells per well in 0.2 ml of culture medium and treated as indicat-

ed in the respective experiments 24h after seeding. 48h after treatment cells were pulsed

with ³H-methylthymidine (0.5lCi) (Amersham Radiochemical Centre, Buckinghamshire, UK)

for another 24h and radionuclide uptake was measured by scintillation counting.

Matrigel invasion assay

Matrigel invasion assay for measuring glioma cell invasion was performed as described pre-

viously (29, 30). For trametinib treatment, a ten-time higher concentration was used com-

pared to proliferation assays as cells were treated only for 24h (vs. 72h in proliferation as-

says). For experiments with transient knockdown of IGFBP1, cells were seeded at 100,000

cells per well in 6-well plates, transfected with siRNA and added to the Boyden chambers

48h after transfection.

Quantitative real-time PCR (qRT-PCR)

RNA extraction, cDNA synthesis and qRT-PCR were performed as previously described

(31). Primer sequences are listed in supplementary table 1.

P53 Sequencing

For p53 sequencing RNA and cDNA of U87MG cells that were a) RG7388 resistant, b)

DMSO control treated and c) wild-type were isolated as previous described (31). Fragments

of exons 2 to 9 of the p53 gene were amplified using PCR technique and sequenced with

Sanger sequencing. Primer sequences are listed in supplementary table 1.

Immunoblot analysis

Whole cell lysates were prepared as described previously (31). The following antibodies

were used: goat IGFBP1 (1:240, R&D-systems, Wiesbaden-Nordenstadt, Germany), mouse




9

phospho-Iκa (1:1,000), rabbit Iκa (1:1,000), rabbit ERK1/2 (1:1,000, all obtained from Cell

Signaling, Cambridge, UK). The PathScan® Multiplex Western Cocktail I (1:200, Cell Signal-

ing) contains antibodies against phospho-ERK1/2, phospho-p90RSK, phospho-AKT (Ser473)

and phospho-S6 ribosomal protein and was mainly used to analyze phospho-ERK1/2. Equal

protein loading was controlled with goat GAPDH (1:5,000; Linaris, Germany) or mouse α-

Tubulin (1:5,000, Sigma Aldrich, USA) staining. For trametinib treatment, a ten-time higher

concentration was used compared to proliferation assays as cells were treated only for 24h

(vs. 72h in proliferation assays).

Flow cytometry

For cell cycle, analysis 100,000 cells were seeded in 6-well plates and treated after 24h with

the indicated concentrations of RG7388 or DMSO control. After 72h cells were incubated in

70% ethanol, stained with 40 µg/ml propidium iodide enriched with 20 µg/ml RNAse and ana-

lyzed in a BD-FACS Canto II flow cytometer. Final data were processed with FloJo flow cy-

tometry analysis software (Treestar). For analysis of apoptosis sub-G1 phase was measured

in cell cycle analysis.

Microarray and gene set enrichment analysis

Microarray analysis was performed by the Genomics and Proteomics Core Facility of the

German Cancer Research Center (Heidelberg, Germany) using Illumina HumanHT-12v4

Expression Bead Chips which analyze the expression levels of 31,000 annotated genes.

Three independent samples of RG7388 resistant and related DMSO control treated U87MG

cells were analyzed for comparison. Data analysis was performed with Ingenuity® Pathway

Analysis (IPA, Ingenuity Systems Inc., Redwood City, USA) and gene set enrichment analy-

sis (GSEA) (32). The GSEA software tool was downloaded from the homepage of the Broad

institute (http://software.broadinstitute.org/gsea). Fifty hallmark gene sets as well as the C2

and C5 gene sets were used for exploratory testing of pathway enrichments. The number of

permutations was set to 1,000.

Statistical analysis




10

Statistical significance was assessed by Student's t-test (Excel, Microsoft, Seattle, WA, USA)

or one-sample t-test as appropriate (Graph Pad Software). P-values of p < 0.05 were con-

sidered significant. Figures represented summaries of at least three independent experi-

ments in proportion to the related control, if not otherwise specified. Immunoblot, qRT-PCR

and flow cytometry data were shown as one representative out of three independent experi-

ments. Quantification of immunoblot data was performed with ImageJ after exclusion of

overexposed blots. Synergistic effects were analyzed based on at least three independent

experiments. Observed inhibition of combination therapy and expected inhibition under inde-

pendence of the individual therapies were calculated using Bliss’ independence method (33).

Synergistic effect of the combination therapy was tested in a linear regression model based

on log-transformed inhibition measurements with main therapy effects, an interaction term

between both therapies and the experiment effect to account for paired measurements as

predictors. An over-additive significant interaction was interpreted as synergism. P-values <

0.05 were considered significant.

Results

Effects of RG7388 treatment in glioblastoma cells

Short-term treatment with 100 nM RG7388 inhibited clonogenicity in p53 wild-type glioblas-

toma cell lines (U87MG and A172, Fig. 1A and Fig. S1A) and p53 wild-type glioma-initiating

cell cultures (GICs; T1, S24, Fig. 1A), whereas p53 mutant (LN18, LN428, U318, U373, Fig.

S1A) and p53 deficient cell lines (LN308) were primary resistant against RG7388 treatment

in a concentration of 100 nM (Fig. S1A). In p53 wild-type glioblastoma cell lines (U87MG,

A172) and GICs (T1, S24), RG7388 led to a significant and dose dependent reduction of

clonogenicity and proliferation (Fig. 1A, Fig. S2, left panel (“no RT”) of each graph). In

U87MG cells, RG7388 induced protein levels of p53 target genes, such as p21 and MDM2

(Fig. S1B), apoptosis (Fig. S1C) and a G1 arrest (Fig. S1D).




11

Synergism of RG7388 treatment and radiotherapy

As radiotherapy is the standard of care for first-line treatment of glioblastoma patients, we

analyzed potential synergistic effects of RG7388 therapy and radiotherapy at clinically rele-

vant radiation doses. Combined treatment of RG7388 in low nanomolar concentrations (10-

100 nM) and radiotherapy at 2 and 4 Gy showed synergistic effects on the inhibition of

clonogenicity (Fig. 1A, middle and right panel, respectively). Calculated expected inhibition of

combined treatment based on impacts of respective monotherapies and observed inhibition

of combined treatment as well as p-values of over-additive interaction are demonstrated in

respective figures to illustrate synergism. As an example, 50 nM RG7388 treatment alone

reduced clonogenicity in S24 cells by 33%, 2 Gy by 32% and 4 Gy by 66%. The combination

of 50 nM RG7388 and 2 Gy inhibited clonogenicity by 86% and in combination with 4 Gy by

94%. Calculated expected inhibition based on effects of respective monotherapies were 68%

for treatment with 50 nM RG7388 combined with 2 Gy radiotherapy and 83% for combination

with 4 Gy. Therefore, these data represented strong synergistic effects of RG7388 treatment

and radiotherapy (both p < 0.001, respectively) (Fig. 1A, S24 cells). These synergistic effects

were substantiated when examining the reduction of proliferation in the p53 wild-type GICs

and glioblastoma cell lines (Fig. S2). Cell viability measurements in freshly isolated murine

astrocytes and neurons did not reveal relevant toxicity of RG7388 monotherapy on murine

normal brain cells in-vitro. While irradiation led to a small, but not relevant, reduction of cell

viability, combined treatment with RG7388 did not further increase toxicity in these cells (Fig.

1B).

Resistance against RG7388 treatment

Resistance against targeted therapies is a main issue in the development of clinically effec-

tive treatments. To analyze possible resistance mechanisms of chronic RG7388 exposure,

RG7388 resistant cells were generated by treating U87MG cells with increasing doses of

RG7388 up to 10 µM over a period of three months. Resistance of these cells against

RG7388 was confirmed by reduced impact on clonogenicity after short-term treatment with




12

RG7388 (Fig. 2A). In addition, RG7388 resistance was maintained in RG7388 resistant cells

after withdrawal of permanent RG7388 exposure for three weeks (Suppl. Fig. 3).

The RG7388 resistant subline showed a more aggressive phenotype: proliferation was 1.5-

times and clonogenicity 2-times increased in RG7388 resistant compared to control cells

(Fig. 2B). Furthermore, resistant cells were 3-times more invasive (Fig. 2B). Short-term

RG7388 treatment over 72h led to an increase of cells in G1 cell cycle phase in long-term

DMSO control treated cells, whereas the RG7388 resistant cells showed a highly increased

amount of cells in G2 phase after three months of RG7388 treatment (Fig. 2C). Interestingly,

resistance was not limited to RG7388 treatment, but radiotherapy was also less effective with

an inhibition of proliferation by 30% at 2 Gy and 56% at 4 Gy in RG7388 resistant cells in

comparison to an inhibition by 45% at 2 Gy and 80% at 4 Gy in DMSO treated control cells

(Fig. 2D).

To identify potential resistance mechanisms, microarray analysis was performed comparing

RG7388 resistant U87MG cells with the respective control cells. Microarray analysis revealed

an activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and nuclear factor kappa-

light-chain-enhancer of activated B cells (NFκB) pathway in RG7388 resistant cells, which

was confirmed by immunoblot analysis (Fig. 2E). Furthermore, among the 20 most regulated

genes in microarray analysis (Table S2) insulin like growth factor binding protein 1 (IGFBP1,

upregulated by 8-fold in RG7388 resistant cells) was the most promising candidate due to

the strongest reliability of confirmation by qRT-PCR (Fig. S6A) and immunoblot analysis (Fig

2E) as well as based on literature research demonstrating a cross-link with the ERK1/2

pathway (34, 35). In addition, microarray analysis showed an activation of the p53 pathway in

RG7388 resistant cells, which was also seen in gene set enrichment analysis (GSEA, Fig.

2F). Of note, p53 sequencing in DMSO control treated and RG7388 resistant cells confirmed

the maintenance of p53 wild-type status in these cells.

Restoring RG7388 sensitivity in RG7388 resistant cells




13

To identify rational treatment strategies to overcome RG7388 resistance, the activated NFκB

and ERK pathways and the enhanced IGFBP1 expression were inhibited. NFκB inhibitor

JSH-23 treatment at 10 µM for 72h (Fig. 3A), but also at up to 30 µM, did not relevantly alter

proliferation as monotherapy. Although combined JSH-23 and RG7388 treatment revealed

significant additive effects on inhibition of proliferation, over-additive effects were not ob-

served as sign for overcoming RG7388 resistance.

Likewise, combining RG7388 and radiotherapy, which was tested based on the synergistic

impact seen in non-resistant cells, did not overcome resistance. Radiotherapy on its own was

slightly anti-proliferative in RG7388 resistant cells, but the addition of RG7388 had no addi-

tional effect (Fig. 3B).

Inhibition of IGFBP1 by transient knockdown via si-RNA revealed synergistic effects in com-

bination with short-term treatment with 100 nM RG7388 in RG7388 resistant U87MG cells.

Although transient IGFBP1 knockdown alone did not relevantly change proliferation of

RG7388 resistant cells, a combined treatment inhibited proliferation by 50% (Fig. 3C). In con-

trast, transient knockdown of IGFBP1 alone significantly ameliorated the increased invasion

in RG7388 resistant cells to the level seen in DMSO control cells (Fig. 3D).

Inhibition of the ERK1/2 signaling pathway by the MEK inhibitor trametinib as monotherapy

had no relevant impact on proliferation of RG7388 resistant U87MG cells in the nanomolar

concentrations tested. However, in contrast to NFκB inhibition and radiotherapy, trametinib

restored sensitivity towards RG7388 therapy with inhibition of proliferation by 51% at 1 nM

trametinib and 61% at 2.5 nM trametinib in combination with 100 nM RG7388 (Fig. 3E). Fur-

thermore, trametinib at 10 nM reduced in particular the pro-invasive phenotype of RG7388

resistant cells leading nearly to normalization of invasiveness when compared to DMSO con-

trol treated cells (Fig. 3F).

Moreover, short-term treatment (for 72h) of both trametinib and RG7388 also showed syner-

gistic effects in A172 and U87MG wild-type cells (Fig. S4).




14

Animal experiments performed with the engineered RG7388 resistant U87MG cells orthotop-

ically implanted in immunodeficient mice confirmed the relevant synergistic effects of com-

bined trametinib and RG7388 treatment at RG7388 resistance (Fig. 4) whereas respective

monotherapy did not significantly reduce growth of these tumors. Comparing changes of tu-

mor volumes in MRI at week 5 after tumor cell implantation (at week 3 after treatment start)

to respective tumor volumes in baseline MRI, tumor growth was inhibited by 9% with

RG7388 monotherapy and 11% with trametinib monotherapy compared to vehicle control

treatment (both not significant). In contrast, combined RG7388 and trametinib treatment re-

duced tumor growth by 67% compared to vehicle control (p=0.012). Moreover, combined

treatment revealed a significant higher reduction of tumor growth compared to RG7388 mon-

otherapy (p=0.01) and trametinib monotherapy (p=0.017). Immunohistochemistry analysis

showed a reduction of phosphor-ERK1/2 after trametinib treatment as demonstration of on-

target efficacy (Suppl. Fig. S5B). Relevant toxicities were not observed with monotherapies

nor combined treatment based on changes in animal weights (Supp. Fig. 5A). The good tol-

erability is further substantiated by available safety data from clinical trials with respective

monotherapies (13, 36, 37).

Resistance is mediated via IGFBP1 - ERK1/2 signaling cascade

When further investigating the signaling pathways affected by the effective treatments de-

scribed, immunoblot analysis revealed that transient knockdown of IGFBP1 reduced not only

IGFBP1 expression but also the activation of ERK1/2 signaling pathway in DMSO control

treated and RG7388 resistant cells (Fig. 5A). Vice versa, short-term treatment with trametinib

reduced IGFBP1 expression in RG7388 resistant cells (Fig. 5B). IGFBP1 binds the IGF re-

ceptor leading to an activation of the ERK1/2 signaling pathway (38). The latter was con-

firmed for the U87MG RG7388 resistant cells. The IGFR inhibitor linsitinib reduced the acti-

vation of ERK1/2 pathway particularly in the U87MG RG7388 resistant cells (Fig. 5C)

In order to further analyze the interaction of IGFBP1 and ERK1/2 signaling on a molecular

level, transcription factors with predicted binding sites at the IGFBP1 promotor were




15

searched in the transcription factor target gene data base (39) and screened for an upregula-

tion in RG7388 resistant cells based on microarray data. A significant upregulation in

RG7388 resistant compared to DMSO control cells was validated for the transcription factors

ZIC2 and NR2F1 by qRT-PCR. In addition, expression of ZIC2 and NR2F1 was significantly

reduced by trametinib treatment (Fig. 5D) as well as by knockdown of IGFBP1 (Fig. 5E).

Knockdown of IGFBP1 and efficacy of trametinib treatment was verified by a reduction of

IGFBP1 expression in RG7388 resistant cells by qRT-PCR (Suppl. Fig S7). Furthermore,

TP53 was also found to have a predicted binding site at the IGFBP1 promotor. Short-term

RG7388 treatment increased IGFBP1 mRNA expression, but did not result in a relevant in-

crease of IGFBP1 protein expression and further activation of ERK1/2 signaling (Fig. 5F). In

contrast, the previous shown stronger activation of the p53 pathway at RG7388 resistance

led to a significant upregulation of IGFBP1 protein expression (Fig. 2E) resulting in an activa-

tion of ERK1/2 signaling and upregulation of the transcription factors ZIC2 and NR2F1 which

then might further enhance the IGFBP1 expression though the binding sites at the IGFBP1

protomor. This self-activating pathway could be inhibited by trametinib treatment or IGFBP1

knockdown, which both resulted in reduced IGFBP1 expression, reduced activation of

ERK1/2 and reduced expression of the transcription factors ZIC2 and NR2F1 (Fig. 5G).

IGFBP1 is strongly upregulated in RG7388 resistant U87MG cells, but basal expression lev-

els in glioblastoma cell lines and GICs are relatively low (Fig. S6B). Therefore, a stable

IGFBP1 overexpression (OE) was induced in the p53 wild-type glioblastoma cell lines

U87MG and A172 to re-evaluate the results in other cells. IGFBP1 overexpression was con-

firmed by immunoblot analysis (Fig. 6A) and resulted in a higher resistance against short-

term RG7388 treatment compared to vector control transfected cells (Fig. 6B), which could

be restored by transient knockdown of IGFBP1 (Fig. 6C). In accordance with the data on

long-term treatment with RG7388 in U87MG cells (“RG7388 resistant cells”), trametinib

treatment also showed synergistic effects in combination with 100 nM RG7388 in IGFBP1

overexpressing cells (Fig. 6D).




16

Discussion

Primary and acquired resistances are a big challenge and limitation for the effective clinical

use of targeted therapies. Whereas primary resistance mechanisms are widely studied for

MDM2 inhibitors in different tumor entities (7, 17, 18, 40), acquired resistance mechanisms

are still not fully understood and were not in the focus in neuro-oncology so far.

Regarding primary resistance, the in-vitro data reconfirm that p53 wild-type status is a pre-

requisite for sensitivity to RG7388 treatment in glioblastoma. Conclusively, these data further

support the use of p53 mutation status as a biomarker for primary response to treatment with

MDM2 inhibitors. Furthermore, RG7388 acts synergistically with radiotherapy in p53 wild-

type glioblastoma cell lines and primary GICs. Combined treatment did not demonstrate rele-

vant effects in freshly isolated murine neurons and astrocytes, which were used to analyze

possible off-target toxicity. The lack of impact compared to tumor cells might be due to the

low proliferation rate and low clonogenicity of neurons and astrocytes. There is no reason to

expect toxicity in resting human cells as well. In conclusion, these preclinical data warrant

further clinical development to study the combination of MDM2 inhibition and radiotherapy as

first-line therapy in glioblastoma patients harboring a p53 wild-type status (41). In addition,

the data validate and relevantly extend the previous stated enhancement of radiosensitivity

with nulin-3a in U87MG glioblastoma cells (19).

Regarding acquired resistance mechanisms, previous studies of different MDM2 inhibitors in

other tumor entities strongly indicate that resistant cells harbor p53 mutations, which are ac-

quired during long-term treatment with MDM2 inhibitors (21-24). In contrast to these data,

resistance of RG7388 long-term treated U87MG glioblastoma cells was not mediated via

acquisition of p53 mutations. Microarray and gene enrichment analysis demonstrated an

activation of p53 pathway in RG7388 resistant compared to DMSO control treated cells. Fur-




17

thermore, microarray analysis revealed an upregulation of IGFBP1 expression as well as an

activation of ERK1/2 and NFκB pathway in RG7388 resistant cells.

Inhibition of NFκB pathway demonstrated additive effects in combination with RG7388 but

did not restore sensitivity towards RG7388 treatment. In contrast, inhibition of ERK1/2 path-

way by the MEK inhibitor trametinib dose-dependently overcame RG7388 resistance and

reduced the highly invasive phenotype of resistant cells. In-vivo experiments further substan-

tiated the relevant benefit of combined RG7388 and trametinib treatment as salvage therapy

at RG7388 resistance demonstrated by a significant higher reduction of tumor growth with

combined treatment compared to respective monotherapies and vehicle control treated mice.

In clinical practice, treatment is often stopped after the emergence of resistance and re-

placed by another salvage therapy. However, the significant synergistic effects of combined

treatment strongly suggest that RG7388 treatment may be continued and the combination

with trametinib should be further explored. Of note, also short-term trametinib and RG7388

treatment was synergistic in A172 and U87MG wild-type cells. Hata et al. demonstrated syn-

ergistic effects of MDM2 and MEK inhibition in KRAS mutant non-small cell lung cancer and

colorectal cancer as first-line therapy (24). Therefore, it remains to be determined if com-

bined first-line treatment may delay RG7388 resistance or results in heterogeneous re-

sistance to one or both of the targeted compounds (24).

Inhibition of IGFBP1 expression by transient knockdown normalized increased invasiveness

in RG7388 resistant cells and reduced proliferation in combination with RG7388 in a syner-

gistic manner. Studies in hepatocytes and colon carcinoma cells suggested a crosslink be-

tween IGFBP1 and ERK1/2 signaling (34, 35), which was confirmed in our cells. Trametinib

treatment reduced IGFBP1 expression and vice versa, transient IGFBP1 knockdown inhibit-

ed ERK1/2 pathway activation. On a molecular level, mRNA expressions of ZIC2 and NR2F1

as candidates for transcription factors with binding sites at the IGFBP1 promotor were up-

regulated in RG7388 resistant cells and inhibited by trametinib treatment and IGFBP1

knockdown. These data further confirm the cross-link between IGFBP1 and ERK1/2 signal-




18

ing and demonstrate a self-activating pathway as a bypass resistance signaling. Short-term

treatment with RG7388 led to an upregulation of IGFBP1 mRNA expression probably via the

TP53 binding site at the IGFBP1 promotor, but did not result in a relevant upregulation of

IGFBP1 protein expression and further activation of ERK1/2 signaling. Upon RG7388 re-

sistance, the TP53 pathway was stronger activated compared to control cells, which resulted

in a higher induction of IGFBP1 mRNA and protein expression and further activation of

ERK1/2 pathway probably via the IGF receptor as inhibition of IGFR reduced ERK1/2 signal-

ing activation particularly in the RG7388 resistant cells. Activation of the IGFBP1 – ERK1/2

signaling further increased the expression of the transcription factors ZIC2 and NR2F1,

which then might increase the IGFBP1 expression via the binding sites at IGFBP1. This self-

activating pathway could be inhibited by trametinib treatment as well as by IGFBP1 knock-

down resulting in a reduction of proliferation and invasion in RG7388 resistant cells. Fur-

thermore, the cross-link to the p53 pathway could explain the synergistic effects of RG7388

and trametinib treatment or IGFBP1 knockdown. After inhibition of the IGFBP1 – ERK1/2

pathway by the treatments described additional RG7388 treatment might again be able to

mainly activate the p53 pathway resulting in an effective reduction of proliferation and inva-

sion at RG7388 resistance.

IGFBP1 expression is generally low in glioblastoma cells but highly upregulated in RG7388

resistant cells. Interestingly, exogenous overexpression of IGFBP1 in A172 and U87MG re-

sulted in development of a more resistant phenotype towards RG7388. In accordance with

the previous described data in RG7388 resistant U87MG cells, combined treatment with ei-

ther trametinib or transient knockdown of IGFBP1 restored sensitivity towards RG7388 ther-

apy in IGFBP1 overexpressing cells. Therefore, these data further confirm the activation of

the ERK1/2 – IGFBP1 signaling cascade as a key mechanism for resistance against

RG7388.




19

In contrast to the synergistic effects of radiotherapy and RG7388 treatment at first-line thera-

py, radiotherapy was not effective in RG7388 resistant cells making re-irradiation as salvage

therapy less attractive.

Long-term treatment often results in selection pressure for more aggressive tumor cells caus-

ing problems for effective salvage therapies. In this study, RG7388 resistant cells showed a

more aggressive phenotype than related control cells with a higher clonogenicity, prolifera-

tion and invasiveness demonstrating the importance for rational, effective and for clinical use

suitable salvage therapies.

In summary, this study presents a mechanism of acquired resistance against MDM2 inhibi-

tors in glioblastoma suggesting rationales for salvage therapies which should be evaluated in

clinical practice. In p53 wild-type glioblastoma cells, RG7388 resistance was mediated via

activation of the ERK1/2 – IGFBP1 signaling cascade, which was effectively targetable by

the clinical approved MEK inhibitor trametinib. The data demonstrated a relevant benefit of

combined trametinib and RG7388 treatment at RG7388 resistance implying that RG788

therapy should be continued and combined with trametinib rather than discontinued after

resistance against RG7388 has occurred. Furthermore, in view of the relevant synergistic

effects, the data further support the combination of radiotherapy and RG7388 treatment in

first-line therapy especially in a situation, when temozolomide as the standard alkylating drug

is of no value because of O6-methylguanine DNA-methyltransferase promoter methylation,

while re-irradiation seems not to be an effective salvage therapy after development of

RG7388 resistance. RG7388 belongs to the targeted therapies evaluated in the ongoing

NCT Neuro Master Match (N2M2) phase I/IIa clinical trial (NCT03158389), which intends to

personalize treatment options based on molecular profiling for glioblastoma patients with an

unmethylated MGMT promotor (41). This is an ideal opportunity to investigate whether the

described mechanism of resistance can be substantiated and the proposed salvage thera-

pies holds true in relapsing glioblastoma after RG7388 treatment.




20

References

1. Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, et al. The somatic genomic landscape of glioblastoma. Cell. 2013 Oct 10;155(2):462-77. 2. Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification data-base. Nucleic Acids Res. 1998 Aug 01;26(15):3453-9. 3. Li Q, Zhang Y, El-Naggar AK, Xiong S, Yang P, Jackson JG, et al. Therapeutic efficacy of p53 restoration in Mdm2-overexpressing tumors. Mol Cancer Res. 2014 Jun;12(6):901-11. 4. Ray-Coquard I, Blay JY, Italiano A, Le Cesne A, Penel N, Zhi J, et al. Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study. Lancet Oncol. 2012 Nov;13(11):1133-40. 5. Andreeff M, Kelly KR, Yee K, Assouline S, Strair R, Popplewell L, et al. Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia. Clin Cancer Res. 2016 Feb 15;22(4):868-76. 6. Vilgelm AE, Pawlikowski JS, Liu Y, Hawkins OE, Davis TA, Smith J, et al. Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apop-tosis and immune clearance of tumor cells. Cancer Res. 2015 Jan 01;75(1):181-93. 7. Verreault M, Schmitt C, Goldwirt L, Pelton K, Haidar S, Levasseur C, et al. Pre-clinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 01;22(5):1185-96. PubMed PMID: 26482041. 8. Shangary S, Wang S. Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res. 2008 Sep 01;14(17):5318-24. PubMed PMID: 18765522. 9. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 2004 Feb 06;303(5659):844-8. 10. Vassilev LT. MDM2 inhibitors for cancer therapy. Trends Mol Med. 2007 Jan;13(1):23-31. 11. Ding Q, Zhang Z, Liu JJ, Jiang N, Zhang J, Ross TM, et al. Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development. J Med Chem. 2013 Jul 25;56(14):5979-83. 12. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, et al. Struc-ture of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation do-main. Science. 1996 Nov 08;274(5289):948-53. 13. Yee KM, G.; Vey, N.; Dickinson, M.; Seiter, K.; Assouline, S.; Drummond, M.; Yoon, S.-S.; Kasner, M.;Lee, J.-H.; Kelly, K; Blotner, S.; Higgins, B.; Middleton, S.; Nichols, G.; Chen, G.; Zhong, H.; Pierceall, W.; Zhi, J.; Chen, L.-C. Phase 1/1b Study of RG7388, a Potent MDM2 Antagonist, in Acute Myelogenous Leukemia (AML) Patients (Pts). Blood. 2014 (124(21):116.). 14. Weller M, van den Bent M, Tonn JC, Stupp R, Preusser M, Cohen-Jonathan-Moyal E, et al. European Association for Neuro-Oncology (EANO) guideline on the di-agnosis and treatment of adult astrocytic and oligodendroglial gliomas. Lancet Oncol. 2017 Jun;18(6):e315-e29. 15. Wick W, Weller M, van den Bent M, Sanson M, Weiler M, von Deimling A, et al. MGMT testing--the challenges for biomarker-based glioma treatment. Nat Rev Neurol. 2014 Jul;10(7):372-85. 16. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005 Mar 10;352(10):997-1003.




21

17. Saddler C, Ouillette P, Kujawski L, Shangary S, Talpaz M, Kaminski M, et al. Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia. Blood. 2008 Feb 01;111(3):1584-93. 18. Lakoma A, Barbieri E, Agarwal S, Jackson J, Chen Z, Kim Y, et al. The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neu-roblastoma. Cell Death Discov. 2015;1. 19. Villalonga-Planells R, Coll-Mulet L, Martinez-Soler F, Castano E, Acebes JJ, Gimenez-Bonafe P, et al. Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme. PLoS One. 2011 Apr 05;6(4):e18588. 20. Tovar C, Rosinski J, Filipovic Z, Higgins B, Kolinsky K, Hilton H, et al. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proc Natl Acad Sci U S A. 2006 Feb 07;103(6):1888-93. 21. Aziz MH, Shen H, Maki CG. Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3. Oncogene. 2011 Nov 17;30(46):4678-86. 22. Jung J, Lee JS, Dickson MA, Schwartz GK, Le Cesne A, Varga A, et al. TP53 mutations emerge with HDM2 inhibitor SAR405838 treatment in de-differentiated lipo-sarcoma. Nat Commun. 2016 Aug 31;7:12609. 23. Hoffman-Luca CG, Ziazadeh D, McEachern D, Zhao Y, Sun W, Debussche L, et al. Elucidation of Acquired Resistance to Bcl-2 and MDM2 Inhibitors in Acute Leuke-mia In Vitro and In Vivo. Clin Cancer Res. 2015 Jun 01;21(11):2558-68. 24. Hata AN, Rowley S, Archibald HL, Gomez-Caraballo M, Siddiqui FM, Ji F, et al. Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers. Oncogene. 2017 Aug 07. 25. Chapeau EA, Gembarska A, Durand EY, Mandon E, Estadieu C, Romanet V, et al. Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf-/- mouse model. Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):3151-6. 26. Lemke D, Weiler M, Blaes J, Wiestler B, Jestaedt L, Klein AC, et al. Primary gli-oblastoma cultures: can profiling of stem cell markers predict radiotherapy sensitivi-ty? J Neurochem. 2014 Oct;131(2):251-64. 27. Silginer M, Burghardt I, Gramatzki D, Bunse L, Leske H, Rushing EJ, et al. The aryl hydrocarbon receptor links integrin signaling to the TGF-beta pathway. Onco-gene. 2016 Jun 23;35(25):3260-71. 28. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparing de-pleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009 Aug 15;347(1-2):70-8. 29. Kessler T, Sahm F, Blaes J, Osswald M, Rubmann P, Milford D, et al. Glioma cell VEGFR-2 confers resistance to chemotherapeutic and antiangiogenic treatments in PTEN-deficient glioblastoma. Oncotarget. 2015 Oct 13;6(31):31050-68. 30. Wick W, Grimmel C, Wild-Bode C, Platten M, Arpin M, Weller M. Ezrin-dependent promotion of glioma cell clonogenicity, motility, and invasion mediated by BCL-2 and transforming growth factor-beta2. J Neurosci. 2001 May 15;21(10):3360-8. 31. Hertenstein A, Schumacher T, Litzenburger U, Opitz CA, Falk CS, Serafini T, et al. Suppression of human CD4+ T cell activation by 3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is mediated by CXCL9 and CXCL10. Biochem Pharmacol. 2011 Sep 15;82(6):632-41. 32. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting ge-nome-wide expression profiles. Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545-50. 33. Bliss CI. The toxicity of poisons applied jointly. Ann Appl Biol 1939;26:585-615. 34. Zhang P, Suidasari S, Hasegawa T, Yanaka N, Kato N. High concentrations of pyridoxal stimulate the expression of IGFBP1 in HepG2 cells through upregulation of the ERK/cJun pathway. Mol Med Rep. 2013 Oct;8(4):973-8.




22

35. Leu JI, Crissey MA, Craig LE, Taub R. Impaired hepatocyte DNA synthetic re-sponse posthepatectomy in insulin-like growth factor binding protein 1-deficient mice with defects in C/EBP beta and mitogen-activated protein kinase/extracellular signal-regulated kinase regulation. Mol Cell Biol. 2003 Feb;23(4):1251-9. 36. Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012 Jul 12;367(2):107-14. 37. Blotner S, Chen LC, Ferlini C, Zhi J. Phase 1 summary of plasma concentration-QTc analysis for idasanutlin, an MDM2 antagonist, in patients with advanced solid tu-mors and AML. Cancer chemotherapy and pharmacology. 2018 Mar;81(3):597-607. 38. Furstenberger G, Senn HJ. Insulin-like growth factors and cancer. Lancet On-col. 2002 May;3(5):298-302. 39. Plaisier CL, O'Brien S, Bernard B, Reynolds S, Simon Z, Toledo CM, et al. Caus-al Mechanistic Regulatory Network for Glioblastoma Deciphered Using Systems Ge-netics Network Analysis. Cell systems. 2016 Aug;3(2):172-86. 40. Coll-Mulet L, Iglesias-Serret D, Santidrian AF, Cosialls AM, de Frias M, Castano E, et al. MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells. Blood. 2006 May 15;107(10):4109-14. 41. Hertenstein A. JD, Sahm F., Pfaff E., Huttner B., Karapanagiotou-Schenkel, A., Eisenmenger A., Osswald M., Wick A., Löw S., Bendszus M., Debus J., Herold-Mende C., Unterber A., von Deimling A., Brors B., Platten M., Pfister S., Wick W. Umbrella pro-tocol for phase I/IIa trials of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed glioblastoma without MGMT promoter methylation Neuro Master Match (N2M2). ASCO Meeting Abstracts, IL: abstract TPS2084. 2016.

Figure legends

Figure 1: Synergistic effects of combined RG7388 treatment and radiotherapy on inhi-

bition 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 com-

bined RG7388 treatment and radiotherapy. *: p<0.05, **: p<0.01, ***: p<0.001.




23

Figure 2: Long-term RG7388 treatment resulted in resistance against RG7388 in

U87MG cells (“RG7388 resistant cells”). A) Short-term RG7388 treatment relevantly re-

duced 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 clono-

genic and more invasive than DMSO treated control cells. C) While short-term treatment with

RG7388 for 72h led to an increase of cells in G1 cell-cycle phase, long-term RG7388 treat-

ment 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 activa-

tion of ERK1/2 and NFκB pathway and an upregulation of IGFBP1 in RG7388 resistant

U87MG cells. F) Gene set enrichment analysis (GSEA) revealed a higher activation of p53

pathway in RG7388 resistant cells compared to DMSO control cells. Figures represent mean

values of at least three independent experiments. Only immunoblot and cell cycle data rep-

resent 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.

Figure 3: Restoring RG7388 sensitivity in RG7388 resistant U87MG cells. A+B) Neither

inhibition of NFκB pathway by 10 µM 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 proliferation in combination with RG7388 treatment. F)

Trametinib treatment reduced the invasive phenotype of RG7388 resistant cells. Due to

shorter treatment compared to proliferation assays (24h vs. 72h) higher concentrations of

trametinib were used in migration assay. Figures represent mean values of at least three




24

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

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 implanta-

tion 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 upper 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

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 con-

trol 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 re-

sistant cells. C) Inhibition of IGF receptor by the IGFR inhibitor linsitinib reduced ERK1/2 sig-

naling activation particularly in U87MG RG7388 resistant cells. D, 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 knock-

down (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 mo-

lecular 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 fur-




25

ther 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

Figure 6: RG7388 resistance is mediated via activation of the IGFBP1 – ERK1/2 signal-

ing pathway: A+B) Exogenous IGFBP1 overexpression (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+D) Short-term

RG7388 treatment combined with transient knockdown of IGFBP1 (C) or in combination with

short-term trametinib treatment (D) showed significant synergistic effects in IGFBP1 overex-

pressing A172 and U87MG cells. Figures represent mean values of at least three independ-

ent experiments. * demonstrates levels of significance calculated between RG7388 treatment

and related DMSO control, ° indicates levels of significance of synergistic effects of com-

bined treatment. *: p<0.05, **: p<0.01, ***: p<0.001. OE: overexpression; tram.: trametinib




Published OnlineFirst October 1, 2018.Clin Cancer Res Anne Berberich, Tobias Kessler, Carina M. Thomé, et al. glioblastoma cells by the MEK inhibitor trametinibTargeting resistance against the MDM2 inhibitor RG7388 in

Updated version

10.1158/1078-0432.CCR-18-1580doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2018/09/29/1078-0432.CCR-18-1580.DC1

Access the most recent supplemental material at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/early/2018/09/29/1078-0432.CCR-18-1580To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-18-1580

http://clincancerres.aacrjournals.org/content/suppl/2018/09/29/1078-0432.CCR-18-1580.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2018/09/29/1078-0432.CCR-18-1580


targeting resistance against the mdm2 inhibitor rg7388 in ......sep 29, 2018 · dysregulation of...

Documents