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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 Jul 30;176(18):3681–3694. doi: 10.1111/bph.14773

Bcl‐2/Bcl‐xL inhibition predominantly synergistically enhances the anti‐neoplastic activity of a low‐dose CUSP9 repurposed drug regime against glioblastoma

Marc‐Eric Halatsch 1, Richard Eric Kast 2, Annika Dwucet 1, Michal Hlavac 1, Tim Heiland 1, Mike‐Andrew Westhoff 3, Klaus‐Michael Debatin 3, Christian Rainer Wirtz 1, Markus David Siegelin 4, Georg Karpel‐Massler 1,✉
PMCID: PMC6715605  PMID: 31222722

Abstract

Background and Purpose

Drug repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti‐cancer activity. In this study, we examined whether inhibition of the anti‐apoptotic Bcl‐2 family proteins Bcl‐2 and Bcl‐xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma.

Experimental Approach

We applied 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide assays to assess cellular proliferation. Annexin V/propidium iodide and tetramethylrhodamine, ethyl ester staining were used to examine apoptosis. Western blotting, RT‐PCR, and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action.

Key Results

Bcl‐2/Bcl‐xL inhibition exerted synergistic anti‐proliferative effects across established, primary cultured, and stem‐like glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its proposed original concentration (CUSP9‐LD). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9‐LD counteracted ABT263‐mediated up‐regulation of Mcl‐1. Silencing of Mcl‐1 enhanced ABT263‐mediated apoptosis which indicates that down‐regulation of Mcl‐1 is crucial for the induction of cell death by the combination treatment.

Conclusion and Implications

These data suggest that Bcl‐2/Bcl‐xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl‐2/Bcl‐xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.

Abbreviations

MTT

3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide

PI

propidium iodide

TMRE

tetramethylrhodamine, ethyl ester

What is already known

  • CUSP9 at full dose is being tested in a clinical trial in recurrent glioblastoma patients.

What this study adds

  • This study adds mechanistic insight into how CUSP9 at low dose works and how to improve its efficacy.

What is the clinical significance

  • This study provides a rationale to improve the efficacy and reduce the toxicity of CUSP9.

1. INTRODUCTION

Primary brain tumours such as glioblastoma are still very difficult to treat because a complete surgical resection in a biological sense is not possible and adjuvant therapies are strongly opposed by the disease (Stupp et al., 2005). A high intratumoural genetic and epigenetic variation among cells that are situated in a strongly protected environment represents a major obstacle for chemotherapeutic agents (Patel et al., 2014). Therefore, the development of novel therapeutic agents is urgently needed but is also highly expensive and time‐consuming to finally reach clinical application.

Drug repurposing is a strategy to safely accelerate discovery of new cancer treatments (reviewed specifically for glioblastoma in Basso, Miranda, Sousa, Pais, & Vitorino, 2018; Seliger & Hau, 2018). Cancer generally and glioblastoma specifically use pre‐existing physiological pathways of growth, anti‐apoptosis, and cell migration, albeit subverted and dysregulated to generate malignancy. Modern medicine has a wide variety of drugs to treat non‐malignant disease that happen to interfere with these pathological, malignancy‐associated, subverted and distorted but otherwise normal pathways. Repurposed drugs are approved by national regulatory authorities, are generally inexpensive, and have well‐known side effect and safety profiles.

CUSP9 represents a therapeutic regimen which comprises nine repurposed drugs with ancillary attributes that interfere with previously identified dysregulated growth and anti‐apoptosis pathways in glioblastoma (Kast, Karpel‐Massler, & Halatsch, 2014). It is currently investigated in a Phase I clinical trial in recurrent glioblastoma (NCT02770378) at our institution and includes the antiemetic aprepitant (80 mg, 1× per day), the antibiotic minocycline (100 mg, 2× per day), the anti‐alcohol abuse drug disulfiram (250 mg, 2× per day), the anti‐inflammatory celecoxib (400 mg, 2× per day), the antidepressant sertraline (100 mg, 2× per day), the anti‐hypertensive captopril (50 mg, 2× per day), the antifungal itraconazole (200 mg, 2× per day), the antiretroviral ritonavir (400 mg, 2× per day), and the anti‐rheumatic auranofin (3 mg, 2× per day) and is accompanied by the standard chemotherapeutic agent temozolomide at a low dose (20 mg·m−2, 2× per day).

The Bcl‐2 family of proteins consists of a molecular network regulating the intrinsic pathway of apoptosis (Adams & Cory, 2007). They consist of pro‐ and anti‐apoptotic members which under normal circumstances are kept in tight balance. In cancer, anti‐apoptotic Bcl‐2 family proteins such as Bcl‐2, Mcl‐1, and Bcl‐xL are frequently overexpressed, which shifts the cellular homeostasis towards an anti‐apoptotic cellular phenotype facilitating the survival of cancer cells. BH3 mimetics like ABT263 (navitoclax) interfere with the function of anti‐apoptotic Bcl‐2 family proteins Bcl‐2 and Bcl‐xL by inhibiting the sequestration of pro‐apoptotic multi‐domain effector proteins BAX and BAK which leads to pore formation in the outer mitochondrial membrane and to restoration of a pro‐apoptotic cellular phenotype (Karpel‐Massler, Ishida, Zhang, Halatsch, et al., 2017; Tse et al., 2008). Moreover, BH3 mimetics induce a displacement of BH3‐only proteins such as NOXA, BAD, or Bim from anti‐apoptotic Bcl‐2 family proteins to further promote apoptosis (Kale, Osterlund, & Andrews, 2018). ABT263 has been investigated in clinical trials for the treatment of lymphoid malignancies or small cell lung cancer (Kipps et al., 2015; Roberts et al., 2015; Rudin et al., 2012; Tolcher et al., 2015; Wilson et al., 2010).

In this study, we show for the first time that CUSP9, when reduced to very low dosage at one tenth to one twentieth of its originally proposed dosage (CUSP9‐LD), sensitizes for intrinsic apoptosis and induces mostly synergistic cell death when combined with the BH3 mimetic ABT263. On the molecular level, CUSP9‐LD down‐regulates Mcl‐1 to circumvent resistance towards ABT263. Overall, our data suggest that an extension of CUSP9 by BH3 mimetics might prove beneficial and allow for further dose reduction to avoid toxic side effects.

2. METHODS

2.1. Cell cultures and growth conditions

U87MG (RRID:CVCL_0022) and A172 (RRID:CVCL_0131) human glioblastoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). T98G (RRID:CVCL_0556) and U251 (RRID:CVCL_0021) human glioblastoma cell lines were purchased from Sigma Aldrich (St. Louis, MO, USA). The identities of the glioblastoma cell lines we purchased were confirmed by the respective source of purchase. The initial stocks were expanded, frozen, and stored in liquid nitrogen. Fresh aliquots were thawed every 6 weeks.

For human tumour samples, consent was obtained from the patient or next of kin and procedures were carried out with the approval of the internal review board. ULM‐GBM‐PC35, ULM‐GBM‐PC38, and ULM‐GBM‐PC40 are primary cultured human glioblastoma cells derived from tumour resections performed at our institution and were generated and characterized as previously described (Karpel‐Massler et al., 2013; Opel et al., 2008; Schneider et al., 2016; Ströbele et al., 2015). ULM‐GBM‐SC35, ULM‐GBM‐SC38, and ULM‐GBM‐SC40 are stem‐like glioma cells which were also derived and characterized at our institution (Karpel‐Massler et al., 2013; Opel et al., 2008; Schneider et al., 2016; Ströbele et al., 2015). All cells were cultured as previously described (Karpel‐Massler, Horst, et al., 2016; Karpel‐Massler, Ishida, Bianchetti, Zhang, et al., 2017). The experiments were carried out under serum starvation (1.5% FBS) to mimic the nutrient‐deprived nature of the micro‐environment to which the majority of glioblastoma cells are exposed to within an actual tumour.

2.2. Cell viability assays

In order to examine cellular proliferation, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) assays were performed as previously described (Karpel‐Massler, Ba, et al., 2015; Karpel‐Massler, Kast, et al., 2015).

2.3. Measurement of apoptosis and mitochondrial membrane potential

Apoptosis was detected by Annexin V/propidium iodide (PI) or PI staining followed by flow cytometry as described before (Karpel‐Massler et al., 2014; Karpel‐Massler, Ramani, et al., 2016). Briefly, 3 × 104 cells were seeded in duplicate on 12‐well plates and allowed to attach overnight. After the respective treatments, the supernatants and enzymatically detached cells (Trypsin/EDTA, Biochrom AG, Berlin, Germany) were collected. The cells were centrifuged and washed twice with ice‐cold Annexin V binding buffer containing 140‐mM NaCl, 2.5‐mM Ca2+, and 1‐M 4‐(2‐hydroxyethyl)piperazine‐1‐ethanesulfonic acid (pH 7.4) followed by resuspension in 100‐μl binding buffer and incubation with 2.5‐μl Annexin‐V‐FLUOS (Roche Diagnostics, Indianapolis, IN, USA) for 15 min at room temperature. Afterwards, the cells were washed once with ice‐cold Annexin V binding buffer prior to resuspension in 300‐μl buffer. PI was added to a final concentration of 2.5 μM right before performing each single measurement. For each flow cytometric analysis, 10,000 events were recorded using the flow cytometer FACSCantoTM II (BD Biosciences, NJ, USA). The BD FACS DIVA software or FCSalyzer software version 0.9.15 was used for further quantitative analysis.

PI staining was performed as described before (Karpel‐Massler, Banu, et al., 2016; Karpel‐Massler, Ishida, Bianchetti, Shu, et al., 2017). Briefly, cells were enzymatically detached and centrifuged for 5 min at 1,800 rpm prior to washing twice with PBS. Then 200‐μl PI staining solution containing 0.05% trisodium citrate dihydrate (Carl Roth, Karlsruhe, Germany), 0.05% Triton‐X 100, and 0.05 mg·ml−1 of PI (Sigma Aldrich) was added, and cells were incubated for 30 min at 4°C prior to flow cytometric analysis. To detect intrinsic apoptosis, staining with tetramethylrhodamine, ethyl ester (TMRE) was performed. Cell culture plates were centrifuged at 317× g for 5 min prior to aspiration of the supernatant and addition of 200‐nM TMRE (Thermo Fisher Scientific, Eugene, OR, USA) staining solution and incubation for 20 min at 37°C. Then cells were enzymically detached and centrifuged for 5 min at 609× g. The supernatant was discarded, and the cells were resuspended in 300‐μl PBS followed by flow cytometric analysis. The data were analysed using the BD FACS DIVA software or FCSalyzer software version 0.9.15.

2.4. Western blot analysis

Specific protein expression in cell lines was determined by western blot analysis as described before (Karpel‐Massler, Horst, et al., 2016; Karpel‐Massler, Ishida, Bianchetti, Zhang, et al., 2017) using the following primary antibodies: Mcl‐1 (RRID:AB_10694494; 1:1,000; #5453S, clone: D35A5, CST: Cell Signaling Technology, Danvers, MA, USA), Bcl‐2 (RRID:AB_394045; 1:1,000; #551098, BD Pharmingen, USA), Bcl‐xL (RRID:AB_630916; 1:500; sc‐1041, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, or RRID:AB_2228008; 1:500; #2764, clone 54H6, CST), human caspase 3 (RRID:AB_331439; 1:1,000; #9662, CST), human caspase 9 (RRID:AB_2068620; 1:1,000; #9508T, clone C9, CST), PARP (RRID:AB_1602926; 1:1,000; #11835238001, Roche, Mannheim, Germany), and β‐actin (RRID:AB_476692; 1:2,000, clone AC15; Sigma Aldrich), and secondary HRP‐linked antibodies were purchased from CST (RRID:AB_330924, #7076S; RRID:AB_2099233, #7074S).

2.5. Transfections of siRNAs

Mcl‐1‐siRNA (ON‐TARGETplus MCL1 siRNA, J‐004501‐16‐0002) and non‐targeting siRNA (ON‐TARGETplus Non‐targeting siRNA, D‐001810‐03‐05) were purchased from Dharmacon (Lafayette, CO, USA). Bcl‐xL‐siRNA I was purchased from Cell Signaling Technology (Signal silence®). Cells were incubated with the formed complexes of Viromer® BLUE (Lipocalyx, Halle, Germany) and the respective siRNA in DMEM with 1.5% FBS for the whole duration of the experiment.

2.6. Real‐time PCR and cDNA synthesis

RT‐PCR was performed as described before (Karpel‐Massler, Ishida, Bianchetti, Shu, et al., 2017) using the primers as outlined below:

Gene Forward sequence Reverse sequence
Mcl‐1 CCA AGA AAG CTG CAT CGA ACC AT CAG CAC ATT CCT GAT GCC ACC T
GAPDH GTC TCC TCT GAC TTC AAC AGC G ACC CTG TTG CTG TAG CCA A
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2.7. Cell migration/motility assays

Scratch assays were performed as previously described (Karpel‐Massler et al., 2013; Karpel‐Massler, Kast, Siegelin, Dwucet, et al., 2017). In brief, subconfluent cell layers in 12‐well plates sustained a scratch across the well carried out with a 200‐μl pipette tip. Sequential microscopic images were taken at defined time points at 10× magnification, and the area of the scratch was further analysed with the NIH ImageJ software (Bethesda, MD; http://imagej.nih.gov/ij, RRID:SCR_003070).

For time‐lapse live cell microscopy imaging, 4 × 104 cells per well were seeded onto 12‐well plates, and microscopic images at 10× magnification were taken with a live‐imaging inverted video microscope (Zeiss Observer.Z1, Göttingen, Germany) every 30 min for a total observation time of 24 hr. During this period, cells were kept at standard culture conditions (37°C, 5% CO2, water‐saturated atmosphere). Single‐cell tracking was performed with the MtrackJ plugin (21) (www.imagescience.org/meijering/software/mtrackj/) for the NIH ImageJ software (http://imagej.nih.gov/ij). Normalized “wind‐rose” plots were generated with the chemotaxis and migration tool from Integrated BioDiagnostics (Ibidi, Martinsried, Germany, www.ibidi.com).

2.8. Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Statistical significance was assessed by one‐way ANOVA and Newman–Keuls post hoc analysis using Prism version 5.04 (GraphPad, La Jolla, CA, RRID:SCR_002798) if not stated otherwise. P < .05 was considered statistically significant.

BLISS analysis was conducted to detect synergistic, additive, or antagonistic effects. The expected total response to the combination treatment was calculated as fractional response to drug A (Fa)+fractional response to drug B (Fb) − Fa × Fb. If the ratio of the actual total response and the expected total response equalled 0.9 to 1.1, additivity was assumed. If this quotient was less than 0.9, the effect was described as antagonistic. Synergism was stated if the quotient was greater than 1.1.

2.9. Materials

Aprepitant (Selleckchem, Houston, TX, USA; stock: 100 mg·ml−1, final concentration.: 0.1 μg·ml−1), auranofin (Sigma Aldrich; stock: 5 mg·ml−1, final concentration : 0.09 μg·ml−1), celecoxib (Sigma Aldrich; stock: 20 mg·ml−1, final concentration : 0.18 μg·ml−1), disulfiram (Sigma Aldrich; stock: 5 mg·ml−1, final concentration : 0.005 μg·ml−1), itraconazole (Abcam, Cambridge, UK; stock: 20 mg·ml−1, final concentration : 0.05 μg·ml−1), ritonavir (Sigma Aldrich; stock: 10 mg·ml−1, final concentration : 1.23 μg·ml−1), temozolomide (Sigma Aldrich; stock: 20 mg·ml−1, final concentration : 0.6 μg·ml−1), ABT199 (Apexbio; stock: 20 mM) and ABT263 (Selleckchem; stock: 10 mM) were dissolved in DMSO. Minocycline (Sigma Aldrich; stock: 25 mg·ml−1, final concentration : 0.25 μg·ml−1), captopril (Sigma Aldrich; stock: 50 mg·ml−1, final concentration : 0.13 μg·ml−1), sertraline (Sigma Aldrich; stock: 3.8 mg·ml−1, final concentration : 0.003 μg·ml−1), and NSC23766 (Calbiochem, EMD Chemicals, Inc., San Diego, CA, USA; stock: 50 mM) were dissolved in H2O. All stock solutions were stored at −20°C. For all experiments, final concentrations of DMSO were below 0.1% (v/v).

2.10. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Fabbro et al., 2017; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. CUSP9‐LD combined with Bcl‐2/Bcl‐xL inhibition has mostly synergistic anti‐proliferative activity

Some of the drugs used in the CUSP9 regimen (see Table 1) are known to inhibit cellular proliferation. To test the hypothesis that anti‐proliferative effects of the CUSP9 regimen at reduced dosage can be enhanced by interference with anti‐apoptotic Bcl‐2 family proteins, established, primary cultured, and stem‐like glioma cells were treated with CUSP9‐LD, ABT263 (Figure 1d), or the combination in the presence or absence of temozolomide. MTT assays were performed to determine anti‐proliferative effects (Figure 1a–c). Combined treatment with CUSP9‐LD and ABT263 resulted for the most part in a synergistic anti‐proliferative effect on all cells tested, as confirmed by BLISS analysis (Tables 2 and 3). Notably, an additional treatment with temozolomide did not add to the anti‐cancer activity of CUSP9‐LD/ABT263 in a statistically significant manner except for ULM‐GBM‐SC35 glioma stem‐like cells.

Table 1.

Chemical structures of the CUSP9 drugs

tabular image

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Figure 1.

Figure 1

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(a) Established, (b) primary cultured, and (c) stem‐like glioma cells were treated for 144 hr under serum starvation (1.5% FBS) as indicated. Cell viability was determined by MTT assay. Data shown are means with SEM from at least four independent experiments. *P < .05, significantly different as indicated; n.s., non‐significant; one‐way ANOVA with Newman–Keuls test. (d) Chemical structure of ABT263. (e) T98G established and ULM‐GBM‐SC40 stem‐like glioma cells were treated for 72 hr as indicated. Representative microphotographs were taken at 40× magnification. White arrows mark cellular blebs

Table 2.

Combined treatment with CUSP9‐LD and ABT263 results in a predominantly synergistic anti‐proliferative effect on established and primary cultured glioblastoma cells

ABT263 0.5 μM 1 μM 2 μM
U87MG 1.781 1.220 1.001
A172 1.383 2.887 1.089
T98G 1.574 1.840 1.067
U251 CUSP9‐LD 0.541 2.284 1.722
ULM‐GBM‐PC35 2.779 1.881 1.196
ULM‐GBM‐PC38 1.566 1.618 1.250
ULM‐GBM‐PC40 1.412 1.286 1.168
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Note. Cells were treated for 144 hr as indicated following MTT assay. BLISS analysis was performed as described in Section 2 to determine synergistic (red), additive (green), or antagonistic effects (blue). Data are representative of four independent experiments.

Table 3.

Combined treatment with CUSP9‐LD and ABT263 results in a predominantly synergistic anti‐proliferative effect on stem‐like glioblastoma cells

ABT263 0.025 μM 0.05 μM 0.1 μM
ULM‐GBM‐SC35 1.342 1.139 1.091
ULM‐GBM‐SC38 CUSP9‐LD 1.229 1.155 1.071
ULM‐GBM‐SC40 1.259 1.166 1.100
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Note. Cells were treated for 144 hr at indicated concentrations following MTT assay. BLISS analysis was performed as described in Section 2 to determine synergistic (red) or additive (green) effects. Note there were no antagonistic effects (blue) identified in this set of experiments. Data are representative of four independent experiments.

3.2. Treatment with CUSP9‐LD sensitizes for apoptosis

Light microscopy revealed that simultaneous treatment with CUSP9‐LD and ABT263 led to morphological changes that were typical of apoptosis and included enhanced cellular fragmentation as well as the formation of cellular blebs (Figure 1e). To further quantify to what extent apoptosis might be involved as a relevant part of the mechanism, staining with Annexin V/PI and flow cytometric analysis were performed. U87MG, T98G established, and ULM‐GBM‐SC40 stem‐like glioblastoma cells that were subjected to treatment with the combination of CUSP9‐LD and ABT263 showed a significantly enhanced fraction of Annexin V‐positive (apoptotic) cells compared to either treatment alone (Figures 2a–d and S1A,B).

Figure 2.

Figure 2

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(a, b) U87MG and T98G glioblastoma cells were treated for 48 hr as indicated under serum starvation (1.5% FBS). Staining with Annexin V/propidium iodide was performed prior to flow cytometric analysis. Representative density plots are shown. Quantitative representation of (c) U87MG and (d) T98G glioblastoma cells treated as described for (a) and (b). Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test . (e) T98G and ULM‐GBM‐PC38 glioblastoma cells were treated for 48 hr with solvent, CUSP9‐LD, 1‐μM ABT263, or the combination under serum starvation (1.5% FBS). Whole‐cell extracts were collected, and Western blot analysis was performed for caspase 3 (C3), cleaved caspase 3 (cC3), caspase 9 (C9), and cleaved caspase 9 (cC9). Actin served as a loading control. Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). (f) T98G glioblastoma cells were treated for 72 hr with CUSP9‐LD/ABT263 in the presence or absence of the pan‐caspase inhibitor zVAD.fmk. Annexin V/propidium iodide staining was performed prior to flow cytometric analysis. Data shown are means with SEM (N = 5). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test. Representative histograms of (g) T98G and (h) ULM‐GBM‐PC38 cells treated for 48 hr with solvent, CUSP9‐LD, 1‐μM ABT263, or the combination prior to staining with TMRE and flow cytometric analysis. (i, j) Quantitative representation of the fraction of cells treated as described for (g) and (h) showing a reduced mitochondrial outer membrane potential (MOMP). Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test

3.3. CUSP9‐LD/ABT263 treatment enhances the activation of initiator and effector caspases

To further verify whether the enhanced pro‐apoptotic effect of the combination therapy is dependent on caspase cleavage, we performed Western blot analyses for the total and cleaved forms of initiator caspase 9 and effector caspase 3 as well as PARP. As shown in Figure 2e, the combination treatment with CUSP9‐LD and ABT263 led to markedly enhanced cleavage of caspases 9 and 3 in T98G established and ULM‐GBM‐PC38 primary cultured glioblastoma cells and to a decreased expression of PARP (Figure S1C). Moreover, co‐treatment with the pan‐caspase inhibitor zVAD.fmk blocked the strong pro‐apoptotic response following the CUSP9‐LD/ABT263 treatment which supports a caspase‐mediated specific apoptotic response as the likely mechanism of action (Figures 2f and S1D).

3.4. Treatment with CUSP9‐LD combined with Bcl‐2/Bcl‐xL activates the intrinsic pathway

ABT263 interferes with the intrinsic pathway of apoptosis. We therefore examined next whether the pro‐apoptotic response towards a combined treatment with CUSP9‐LD and ABT263 is mitochondrially driven. Staining with TMRE was performed to assess treatment‐related changes of the mitochondrial outer membrane potential. T98G and ULM‐GBM‐PC38 cells subjected to the combination therapy showed markedly enhanced mitochondrial outer membrane permeability when compared to cells treated with each compound alone, indicating a strong activation of the intrinsic apoptotic pathway (Figure 2g–j).

3.5. CUSP9‐LD opposes ABT263‐mediated up‐regulation of Mcl‐1

Increased expression of Mcl‐1 is a well‐characterized mechanism of resistance towards ABT263 (Konopleva et al., 2006). Our data show that treatment with ABT263 alone results in increased expression of Mcl‐1 in U87MG and A172 glioblastoma cells (Figure 3a,b). In contrast, cells treated with CUSP9‐LD show a decrease in Mcl‐1 levels when applied alone or in combination with ABT263.

Figure 3.

Figure 3

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(a) U87MG and A172 cells were treated for 72 hr as indicated under serum starvation (1.5% FBS). Whole‐cell extracts were collected, and Western blot analysis was performed for Mcl‐1, Bcl‐2, and Bcl‐xS/L. Actin served as a loading control. The arrow head marks the specific band of Mcl‐1. (b) Quantitative representation of the Mcl‐1 expression in U87MG and A172 cells treated as described for (a). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test. (c) A172 cells were treated with non‐targeting (n.t.)‐siRNA or Mcl‐1‐siRNA in the presence or absence of 1‐μM ABT263. Staining with Annexin V/propidium iodide was performed followed by flow cytometric analysis. Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test. (d) A172 cells were treated as described for (c). Western blot for Mcl‐1 was performed to confirm sufficient knockdown. (e) T98G cells were treated with non‐targeting (n.t.)‐siRNA or Bcl‐xL‐siRNA in the presence or absence of CUSP9‐LD prior to staining with Annexin V/propidium iodide and flow cytometric analysis. Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; one‐way ANOVA with Newman–Keuls test. (f) T98G cells were treated as described for (e). Western blot analysis for Bcl‐xL was performed to confirm sufficient knockdown. (g) T98G glioblastoma cells were treated for 48 hr as indicated under serum starvation (1.5% FBS). Staining with Annexin V/propidium iodide was performed prior to flow cytometric analysis to determine the percentage of viable (non‐apoptotic) cells. Data shown are means with SEM (N = 4). n.s., non‐significant; one‐way ANOVA with Newman–Keuls test. (h) A172 glioblastoma cells were treated for 7 or 48 hr with solvent, CUSP9‐LD, 1‐μM ABT263, or the combination. RNA was isolated, and RT‐PCR was performed for Mcl‐1. Data shown are means with SEM (N = 4). (i) A172 cells were pretreated for 5 hr with solvent or CUSP9‐LD prior to adding 10 μg·ml−1 of cycloheximide. At indicated time points, whole‐cell extracts were collected, and Western blot analysis was performed for Mcl‐1. Densitometric analysis was performed using ImageJ (NIH; http://imagej.nih.gov/ij). (j) U87MG and A172 cells were treated for 72 hr as indicated under serum starvation (1.5% FBS). Whole‐cell extracts were collected, and Western blot analysis was performed for Mcl‐1. Actin served as a loading control. Densitometric analysis was performed using ImageJ (NIH; http://imagej.nih.gov/ij). Data shown are means with SEM (N = 4). n.s., non‐significant; one‐way ANOVA with Newman–Keuls test. (k) U87MG, A172, and T98G glioblastoma cells were treated for 144 hr under serum starvation (1.5% FBS) as indicated. Cell viability was determined by MTT assay. Data shown are means with SEM (N = 4). *P < .05, significantly different as indicated; n.s., non‐significant; one‐way ANOVA with Newman–Keuls test

3.6. Selective inhibition of Mcl‐1 enhances ABT263‐mediated apoptosis

We next assessed whether down‐regulation of Mcl‐1 as observed after treatment with CUSP9 is sufficient to explain the enhanced pro‐apoptotic effect when combined with ABT263. To this purpose, we used specific siRNA to silence Mcl‐1 and observed that Mcl‐1 knockdown sensitized A172 glioblastoma cells to the pro‐apoptotic effects of ABT263 (Figures 3c,d and S2A).

3.7. Specific inhibition of Bcl‐xL enhances apoptosis when combined with CUSP9‐LD

ABT263 targets the anti‐apoptotic Bcl‐2 family proteins Bcl‐2 and Bcl‐xL. We next addressed the question whether inhibition of Bcl‐xL alone suffices to enhance apoptosis when combined with CUSP9‐LD. T98G cells were silenced for Bcl‐xL using specific siRNA and treated either with solvent or CUSP9‐LD (Figures 3e,f and S2B). As shown in Figure 3e, selective knockdown of Bcl‐xL caused a significant increase in the fraction of Annexin V‐positive (apoptotic) cells when combined with CUSP9‐LD. Moreover, treatment with the selective Bcl‐2 inhibitor ABT199 did not lead to an enhanced inhibitory effect on the cellular viability of T98G glioblastoma cells when combined with CUSP9‐LD (Figure 3g).

3.8. CUSP9‐LD down‐regulates Mcl‐1 on a post‐transcriptional level

We next addressed the question how CUSP9‐LD down‐regulates Mcl‐1 on the molecular level. RT‐PCR experiments were performed to examine whether CUSP9‐LD affects transcription of Mcl‐1. As shown in Figure 3h, treatment with CUSP9‐LD alone does not reduce transcript levels of Mcl‐1 to a notable degree in A172 cells after 7 hr. After 48 hr of treatment, a slight decrease was noted which, however, was not statistically significant. Treatment with ABT263 or with CUSP9‐LD/ABT263 caused an increase in Mcl‐1 mRNA levels after 48 hr of treatment. The combination treatment was not associated with a decrease in Mcl‐1 mRNA levels.

We further assessed if the CUSP9‐LD‐mediated reduction in Mcl‐1 protein levels is post‐translationally driven. A172 cells were treated with the protein synthesis inhibitor cycloheximide in the presence or absence of CUSP9‐LD (Figure 3i). The protein stability of Mcl‐1 was markedly decreased in the presence of CUSP9‐LD which indicates that CUSP9‐LD affects Mcl‐1 levels at least in part using a post‐translational mechanism.

Celecoxib has been described to down‐regulate Mcl‐1 levels (Gallouet et al., 2014; Song, Chen, & Xing, 2013). We therefore examined whether celecoxib alone could be responsible for the inhibitory effect on Mcl‐1 expression and the predominantly synergistic anti‐proliferative activity of CUSP9‐LD/ABT263. Treatment with celecoxib alone at the concentration used within CUSP9‐LD did not result in a reduced expression of Mcl‐1 protein (Figure 3j). Moreover, while the combination treatment with CUSP9‐LD and ABT263 led to a markedly enhanced impairment of the cellular viability of U87MG, A172, and T98G glioblastoma cells, combined treatment with celecoxib and ABT263 did not yield such a response (Figure 3k).

3.9. CUSP9‐LD has anti‐migratory activity which can be further enhanced by inhibition of RAC1

We further examined whether treatment with CUSP9‐LD affects the migratory behaviour of glioblastoma cells. In U87MG and ULM‐GBM‐PC38 cells, non‐directed migration was markedly impaired following treatment with CUSP9‐LD (Figures 4a,d and S3A,C). In T98G cells, this effect was not noted; however, these cells are characterized by a lower base line migratory activity (Figure S3B,D). The presence of ABT263 did not enhance migration on its own and also did not enhance the anti‐migratory activity of CUSP9‐LD in a notable manner. As migratory activity promotes therapeutic resistance, we next sought for additional compounds to complement our therapeutic approach in order to allow for better control of migration. RAC1 is necessary for podia formation and therefore important for cellular migration. We next performed experiments extending our combination therapy with the RAC1 inhibitor NSC23766. Combined treatment with CUSP9‐LD, ABT263, and NSC23766 resulted in a significantly enhanced anti‐migratory activity in U87MG, T98G, and ULM‐GBM‐PC38 cells (Figure 4a–f). These findings were confirmed by a second independent method using a scratch assay in ULM‐GBM‐PC38 cells (Figure 4g,h). To address the question of whether the anti‐migratory activity of a combined treatment with CUSP9‐LD, ABT263, and NSC23766 could be secondary to an increased cell death, we determined the percentage of apoptotic cells at the end of the experiments. While there was a slight increase in the percentage of apoptotic cells following the combination treatment, no statistically significant difference was found when compared to control (Figure S3E–G).

Figure 4.

Figure 4

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(a) U87MG, (b) T98G, and (c) ULM‐GBM‐PC38 cells were seeded on 24‐well plates followed by sequential microscopic imaging (magnification, 10×) over a total time period of 24 hr. Single‐cell tracking was performed using the MtrackJ software (see Section 2). Wind‐rose plots displaying the paths of 15 single cells per treatment condition during the 24‐hr observation period are shown. The tracks were aligned to start from the same initial position to facilitate comparisons. (d–f) Total distance of 15 cells covered within 24 hr per treatment condition. Data shown are means with SEM (N = 4). P<.05, significantly different as indicated; one‐way ANOVA with Newman‐Keuls test (d); Student's t test (e, f). (g) Monolayers of subconfluent ULM‐GBM‐PC38 cells were scratched prior to treatment with either solvent or CUSP9‐LD/1‐μM ABT263 (ABT)/10‐μM NSC23766 (NSC). Microscopic images were taken at time zero and 18 hr after infliction of the scratch. Magnification, 10×. (h) ULM‐GBM‐PC38 cells were treated as described for (g). The area of the open scratch was determined after 6, 12, 18, and 24 hr using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data presented are means with SD (N = 4). P<.05, significantly different from treated cells; one‐way ANOVA with Newman–Keuls test

4. DISCUSSION AND CONCLUSIONS

Glioblastoma is, de facto, an incurable disease, and little has changed for more than 50 years with respect to the prognosis of patients with this diagnosis, beyond the introduction of temozolomide (Stupp et al., 2005). The currently applied standard therapeutic concepts in the clinic fail to address the biological and genetic complexity of glioblastoma which involves not only a vast intratumoural heterogeneity but also a tumour–stroma interaction including, for instance, pro‐angiogenic and immunosuppressive effects (Patel et al., 2014). The idea of developing a broad‐spectrum therapeutic approach with low toxicity that simultaneously targets many different central pathways and mechanisms seems tempting.

In this context, drug repurposing provides a means to combine several, already marketed and well‐studied medications with anti‐cancer activity, minimizing risks of toxicity and avoiding costly and time‐consuming routes to clinical application. With this in mind, the strategy of the present study was to examine ways of extending the already conceptualized and repurposed drug cocktail CUSP9 to cut down on drug dosages and increase its anti‐cancer activity. We were able to show a remarkable anti‐neoplastic activity of the CUSP9 regime when reduced up to one twentieth of its original concentration and combined with the Bcl‐2/Bcl‐xL inhibitor ABT263 in the low μM range. In addition, anti‐migratory effects were significantly enhanced when this drug cocktail was further extended by adding a RAC1 inhibitor.

On the molecular level, our data show that CUSP9‐LD by itself caused only a slight down‐regulation of Mcl‐1. However, when combined with ABT263, CUSP9‐LD counteracted ABT263‐driven up‐regulation of Mcl‐1, which constitutes a well‐described mechanism increasing drug response towards ABT263 (Konopleva et al., 2006). It seemed tempting to identify one individual drug out of CUSP9‐LD that would be responsible for the inhibitory effect on Mcl‐1 expression and the predominantly synergistic anti‐neoplastic activity. Based on the literature, celecoxib was a potential candidate (Gallouet et al., 2014; Song et al., 2013). However, at the concentration used within CUSP9‐LD, celecoxib neither decreased expression of Mcl‐1 in our setting nor was it sufficient to achieve similar biological activity when combined with ABT263 alone, compared to the full cocktail. Overall, if one considers the complexity of the proposed therapeutic regimen including nine different drugs plus ABT263, it seems unlikely that only one molecular mechanism is completely responsible for the synergistic activity of CUSP9‐LD and ABT263. Thus, down‐regulation of Mcl‐1 is likely to constitute one molecular event that is part of a reprogramming of multiple signalling pathways resulting in the enhanced anti‐glioblastoma activity we observed.

Whereas in traditional approaches, the standard therapeutic modalities are usually only complemented by one additional compound (Chinot et al., 2014; Gilbert et al., 2014; van den Bent et al., 2009), CUSP9 is unique in that it comprises a combination of nine additional drugs, a strategy that is even further extended by the therapeutic approach proposed in our study. This concept of multi‐targeting is well established in other medical fields, for instance, for the treatment of infectious diseases such as tuberculosis (World Health Organization, 2016b) or human immunodeficiency virus infection (World Health Organization, 2016a) but has not been widely adopted in terms of glioma therapy, so far.

Of course, one needs to be cautious and aware that despite the fact that most repurposed drugs are well studied and the respective side effect profiles are well characterized, when combined as a multi‐drug cocktail, previously unknown drug–drug interactions are possible and potentially pose a risk to patients. For instance, addition of ABT263 is likely to favour thrombocytopenia. Therefore, the treatment of patients with multi‐drug cocktails needs to be closely monitored and continuously adapted to the individual patient's side effects. Moreover, the ability of drugs to penetrate into the tumour tissue located within the brain is another variable that needs to be critically considered when targeting brain tumours. It is currently uncertain whether ABT263 is able to cross the blood–brain or blood–tumour barrier in amounts adequate to attain effective levels in patients. Based on our previous studies using orthotopic murine glioma models, systemic application of ABT263 showed therapeutic efficacy (Karpel‐Massler, Ishida, Bianchetti, Zhang, et al., 2017). In addition, convection‐enhanced delivery represents a means to potentially circumvent this problem and to improve drug delivery to brain tumours. This technique has already been safely applied in brain tumour patients (Jahangiri et al., 2016; Kunwar et al., 2010).

Overall, although this therapeutic strategy cannot be taken to an infinite number of drugs that patients are treated with, it may well lead to a crossing point at which precision medicine unifies with poly‐pharmacology. Current technology provides the means to create an interface that integrates information derived from tumour OMICS analyses and reference databases like the Library of Integrated Network‐Based Cellular Signatures (NIH LINCS) to create an individualized repurposed and multi‐targeting approach. This strategy would incorporate a targeted component by addressing individually altered glioblastoma driver genes in addition to a biomarker‐independent “base cocktail.” Initial steps have been taken in this direction and will, we hope, allow for better tumour control in glioblastoma patients in the future.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

G.K.M. contributed to the conception and design. M.‐E.H., G.K.M., and R.E.K. did the development of methodology. A.D., T.H., and G.K.M. performed the acquisition of data. A.D. and G.K.M. did the analysis and interpretation of data. M.‐E.H., G.K.M., and C.R.W. contributed to the administrative, technical, and material support. K.‐M.D., A.D., M.‐E.H., M.H., G.K.M., R.E.K., M.D.S., M.‐A.W., and C.R.W. did the writing, review, and revision of the manuscript. G.K.M. contributed to the study supervision.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, and Immunoblotting and Immunochemistry, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1: A, ULM‐GBM‐SC40 glioblastoma cells were treated for 48 h as indicated under serum starvation (1.5% FBS). Staining with Annexin V/Propidium iodide was performed prior to flow cytometric analysis. Representative density plots are shown. B, Quantitative representation of ULM‐GBM‐SC40 glioblastoma cells treated as described for A. Column, mean. Bar, SEM. N = 4. **p < 0.01, ***p < 0.001. C, A172 glioblastoma cells were treated for 48 h with solvent, CUSP9‐LD, 1 μM ABT263 or the combination under serum starvation (1.5% FBS). Whole cell extracts were collected and Western blot analysis was performed for PARP. Actin served as a loading control. Densitometric analysis was perfomed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Column, mean. Bar, SEM. N = 4. Statistical significance was assessed by Student's t‐test. D, Representative density plots of T98G glioblastoma cells that were treated for 72 h with CUSP9‐LD/ABT263 in the presence or absence of the pan‐caspase inhibitor zVAD.fmk. Annexin V/Propidium iodide staining was performed prior to flow cytometric analysis.

Click here for additional data file. (2.5MB, tif)

Figure S2: A, A172 cells were treated with non‐targeting (n.t.)‐siRNA or Mcl‐1‐siRNA in the presence or absence of 1 μM ABT263. Staining with Propidium iodide was performed followed by flow cytometric analysis. Representative histograms are shown. B, T98G cells were treated with non‐targeting (n.t.)‐siRNA or Bcl‐xL‐siRNA in the presence or absence of CUSP9‐LD prior to staining with Annexin V/Propidium iodide and flow cytometric analysis. Representative density plots are shown.

Click here for additional data file. (1.3MB, tif)

Figure S3: A‐B, ULM‐GBM‐PC38 (A) and T98G (B) cells were seeded on 24‐well plates followed by sequential microscopic imaging (magnification, ×10) over a total time period of 24 h. Single‐cell tracking was performed using the MtrackJ software (see Materials and Methods). Wind‐rose plots displaying the paths of 15 single cells per treatment condition during the 24‐h observation period are shown. The tracks were aligned to start from the same initial position to facilitate comparison. Data are representative for 4 independent experiments. C‐D, Total distance of ULM‐GBM‐PC38 (C) and T98G (D) cells covered within 24 h per treatment condition. Column, mean. Bar, SEM. N = 4. n.s. = non‐significant. *** = p < 0.001. E, U87MG cells were treated for 24 h with 10 μM NSC23766, 1 μM ABT263 or CUSP9‐LD as indicated. Microscopic imaging (magnification, ×10) was performed and apoptotic cells within 3 high‐power fields were counted. Column, mean. Bar, SEM. N = 4. F‐G, T98G (F) and ULM‐GBM‐PC38 (G) cells were treated for 24 h as indicated. Microscopic imaging (magnification, ×10) was performed and apoptotic cells within 3 high‐power fields were counted. Column, mean. Bar, SEM. N = 4. Statistical significance was assessed by Student's t‐test.

Click here for additional data file. (2.1MB, tif)

ACKNOWLEDGEMENTS

We thank Angelika Vollmer for her help with the time‐lapse analyses. This study was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (K08 NS083732, R01 NS095848, and R01 NS102366) to M.D.S.

Halatsch M‐E, Kast RE, Dwucet A, et al. Bcl‐2/Bcl‐xL inhibition predominantly synergistically enhances the anti‐neoplastic activity of a low‐dose CUSP9 repurposed drug regime against glioblastoma. Br J Pharmacol. 2019;176:3681–3694. 10.1111/bph.14773

Halatsch Marc‐Eric and Kast Richard Eric contributed equally.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: A, ULM‐GBM‐SC40 glioblastoma cells were treated for 48 h as indicated under serum starvation (1.5% FBS). Staining with Annexin V/Propidium iodide was performed prior to flow cytometric analysis. Representative density plots are shown. B, Quantitative representation of ULM‐GBM‐SC40 glioblastoma cells treated as described for A. Column, mean. Bar, SEM. N = 4. **p < 0.01, ***p < 0.001. C, A172 glioblastoma cells were treated for 48 h with solvent, CUSP9‐LD, 1 μM ABT263 or the combination under serum starvation (1.5% FBS). Whole cell extracts were collected and Western blot analysis was performed for PARP. Actin served as a loading control. Densitometric analysis was perfomed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Column, mean. Bar, SEM. N = 4. Statistical significance was assessed by Student's t‐test. D, Representative density plots of T98G glioblastoma cells that were treated for 72 h with CUSP9‐LD/ABT263 in the presence or absence of the pan‐caspase inhibitor zVAD.fmk. Annexin V/Propidium iodide staining was performed prior to flow cytometric analysis.

Click here for additional data file. (2.5MB, tif)

Figure S2: A, A172 cells were treated with non‐targeting (n.t.)‐siRNA or Mcl‐1‐siRNA in the presence or absence of 1 μM ABT263. Staining with Propidium iodide was performed followed by flow cytometric analysis. Representative histograms are shown. B, T98G cells were treated with non‐targeting (n.t.)‐siRNA or Bcl‐xL‐siRNA in the presence or absence of CUSP9‐LD prior to staining with Annexin V/Propidium iodide and flow cytometric analysis. Representative density plots are shown.

Click here for additional data file. (1.3MB, tif)

Figure S3: A‐B, ULM‐GBM‐PC38 (A) and T98G (B) cells were seeded on 24‐well plates followed by sequential microscopic imaging (magnification, ×10) over a total time period of 24 h. Single‐cell tracking was performed using the MtrackJ software (see Materials and Methods). Wind‐rose plots displaying the paths of 15 single cells per treatment condition during the 24‐h observation period are shown. The tracks were aligned to start from the same initial position to facilitate comparison. Data are representative for 4 independent experiments. C‐D, Total distance of ULM‐GBM‐PC38 (C) and T98G (D) cells covered within 24 h per treatment condition. Column, mean. Bar, SEM. N = 4. n.s. = non‐significant. *** = p < 0.001. E, U87MG cells were treated for 24 h with 10 μM NSC23766, 1 μM ABT263 or CUSP9‐LD as indicated. Microscopic imaging (magnification, ×10) was performed and apoptotic cells within 3 high‐power fields were counted. Column, mean. Bar, SEM. N = 4. F‐G, T98G (F) and ULM‐GBM‐PC38 (G) cells were treated for 24 h as indicated. Microscopic imaging (magnification, ×10) was performed and apoptotic cells within 3 high‐power fields were counted. Column, mean. Bar, SEM. N = 4. Statistical significance was assessed by Student's t‐test.

Click here for additional data file. (2.1MB, tif)

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