ANTICANCER RESEARCH 36: 599-610 (2016)

Polo-like Kinase Inhibitor Volasertib Exhibits Antitumor Activity and Synergy with in Pediatric Malignancies

SAMUEL ABBOU1*, CLAUDIA LANVERS-KAMINSKY2*, ESTELLE DAUDIGEOS-DUBUS1, LUDIVINE LE DRET1, CORINNE LAPLACE-BUILHE3, JAN MOLENAAR4, GILLES VASSAL1 and BIRGIT GEOERGER1; within the ITCC Biology and Preclinical Evaluation Committee

1Vectorology and Anticancer Therapies, UMR 8203, CNRS, Paris-Sud University, Gustave Roussy Institute, Paris-Saclay University, Villejuif, France; 2Department of Pediatric Hematology and Oncology, University Children’s Hospital, Muenster, Germany; 3Gustave Roussy Institute, Optical Imaging and Flow Cytometry Platform, UMR8081 - IR4M, Villejuif, France; 4Amsterdam Medical Center, Amsterdam, the Netherlands

Abstract. Background: Polo-like kinase 1 (PLK1) controls the Volasertib showed synergistic activity with vincristine but main cell-cycle checkpoints, suggesting utility of its inhibition antagonistic effects with . Conclusion: These findings for cancer treatment, including of highly proliferative pediatric support the further exploration of volasertib for pediatric cancer. This preclinical study explored the selective PLK1 malignancies, particularly alveolar rhabdomyosarcoma, and its inhibitor volasertib (BI 6727) alone and combined with combination with mitotic . in pediatric malignancies. Materials and Methods: Inhibition of proliferation was explored in vitro using Pediatric cancers exhibit a high growth potential, often with dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium an elevated mitotic index. Specific inhibitors targeting (MTS) assay. Mice bearing human xenografts were treated with involved in and organization are required weekly intravenous injections of volasertib. Results: Volasertib for further therapeutics. Polo-like kinase 1 (PLK1) is a inhibited proliferation in all 40 cell lines tested, with a mean serine/threonine kinase which plays a central role in the cell half-maximal growth inhibitory concentration of 313 nmol/l cycle and is essential for mitosis and genomic stability (1). It (range: 4-5000 nmol/l). Volasertib was highly active against contributes to the regulation of the G2/M checkpoint and RMS-1 alveolar rhabdomyosarcoma xenografts, resulting in mitotic entry (2, 3). During mitosis, PLK1 drives microtubular 100% tumor regression. Activity was associated with complete nucleation and its inhibition causes G2/M arrest (4). PLK1 is and prolonged G2/M arrest and subsequent apoptotic cell death. overexpressed in many types of cancer and has been reported as a prognostic factor in adult cancer (5-8). PLK1 is reportedly expressed at a high level in hepatoblastoma (9) and neuroblastoma, where high PLK1 expression correlated with The work was presented in part at the 24th EORTC-NCI-AACR poor prognosis (10). Symposium on ‘Molecular Targets and Cancer Therapeutics’, 6-9 Given its fundamental role in the , inhibition of November 2012 Dublin, Ireland (#Abstract 253) and was supported by PLK1 has been suggested as a new therapeutic approach in the 6th European Framework programme “KidsCancer Kinome” (KCK) cancer treatment. Antitumor effect in vivo has been observed as part of the LSH-2005-2.2.0-3-Innovative diagnostic approaches and in several adult cancer types (11-13). PLK1 inhibition using novel therapies of childhood cancers LIFESCIHEALTH-6037390. SA was supported by a grant of the Fondation de la Recherche Medicale. siRNA (14) or the specific inhibitor BI 2536 (10, 15) have shown antitumor effects in pediatric rhabdomyosarcoma and *These Authors contributed equally to this study. neuroblastoma. Volasertib (BI 6727) is a selective PLK1 inhibitor with superior pharmacokinetic characteristics further Correspondence to: Birgit Geoerger, MD, PhD, Vectorology and developed for clinical use. Volasertib induces cytotoxicity, Anticancer Therapies, UMR 8203, CNRS, Univ. Paris-Sud, Gustave G2/M arrest and in vitro and antitumor effect in vivo Roussy, Université Paris-Saclay, 94 805 Villejuif, France. Tel: +33 in human colonic carcinoma and non-small cell lung cancer 142114661, Fax: +33 142115245, e-mail: birgit.geoerger@ gustaveroussy.fr models (12). Volasertib as a single agent exhibited potential activity in a large panel of pediatric tumors with no histotype Key Words: Cell-cycle checkpoint, polo-like kinase 1 combination, selectivity (16). The phase I trial of volasertib in pediatric rhabdomyosarcoma, neuroblastoma, childhood tumors. malignancies is ongoing (NCT01971476).

0250-7005/2016 $2.00+.40 599 ANTICANCER RESEARCH 36: 599-610 (2016)

This preclinical study evaluated volasertib in vitro and in calculated by the Chou and Talalay method using CalcuSyn software vivo against various pediatric tumor types. Given its (Biosoft, Cambridge, UK) (19). The combination index (CI) values mechanism of action, volasertib would likely be used in for synergism are 0 to <0.9, for additivity between >0.9 and 1.1, for antagonism >1.1 to infinity. combination. A synergistic induction of apoptosis of BI 2536 with vincristine was recently described in neuroblastoma cells Western blot analysis. Total lysates of adherent and supernatant cells (15). We, therefore, explored the combination of volasertib were separated electrophoretically and immunodetected as previously with the mitotic spindle poison vincristine and topoisomerase described (17) using monoclonal mouse antibody to human β-actin II inhibitor etoposide in order to evaluate the prospect of (AC-15, diluted 1:1000; Sigma-Aldrich, St Quentin-Fallavier, France), clinical development in children with advanced malignancies. and rabbit polyclonal antibodies to human Poly [ADP-ribose] polymerase 1 (PARP1) (Ab-2, 1:600; Calbiochem, Darmstadt, Materials and Methods Germany) and caspase 3 (1:600, Cell Signaling Technology, Ozyme, St Quentin-en-Yvelines, France). . Volasertib (BI 6727; kindly supplied by Dr Dorothea Rudolph, Cell-cycle analysis by flow cytometry. Cells were treated in Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria) was exponential growth phase with volasertib at 10, 30 and 100 nM, stored as powder protected from light at room temperature. For in harvested at 2, 6, 18, 24, 48 and 72 h, stained with propidium iodide vitro experiments, volasertib was dissolved in a stock solution of buffer and DNA content was measured using a FACScalibur (BD dimethyl sulfoxide (DMSO) and Roswell Park Memorial Institute Biosciences, Erembodegem, Belgium) (20). (RPMI) 1640 medium to a concentration of 1 mg/ml. For in vivo experiments, powder was resuspended in 0.9% NaCl with 0.1 N HCl In vitro imaging. Cells were seeded in a 35 mm Ibidi petri μ-dish to a final concentration of 3 and 4 mg/ml. Vincristine and etoposide (Biovalley, Marne la Vallée, France) and incubated after 24 hours with were purchased (Teva, Petach Tikva, Israel). volasertib at 100 nM. Cell cytoplasm was stained with 1 μmol carboxy-fluorescein diacetate succinimidyl ester (CFSE) (Molecular Cell lines. The Innovative Therapies for Children with Cancer (ITCC) Probes, Eugene, OR, USA) for 1 minute and cell DNA was stained cell line panel (http://www.itcc-consortium.org/targeted-drug- with 25 nM SYTO® 59 (Molecular Probes). Video was acquired, evaluation.php) comprised 40 cell lines of six pediatric tumor types: starting immediately after volasertib addition, with a FluoView FV10i five medulloblastoma (DAOY, D283 MED, D341 MED, MED-MEB- confocal microscope (Olympus, Hamburg, Germany) at 492 (CFSE - 8A, UW228.2), and seven each of Ewing sarcoma [A673, EW 7, green) and 622 nm (SYTO® 59 - red) over 10 hours; one image per ORS, POE, RD-ES, SIM(EW27), STA-ET-1], acute lymphoblastic hour was captured. leukemia (ALL) (HpB-ALL, KOPN-8, MOLT-16, REH, SEM, SUP- B15, UOC-B6), neuroblastoma (IMR-32, NGP, SH-SY5Y, SJ-NB-6, Gene-expression profiling. RNA expression of HG-U133 Plus 2.0 SJ-NB-8, SK-N-AS, SK-N-BE), osteosarcoma (HOS, IOR-OS-9, microarrays (Affymetrix, Santa Clara, CA, USA) were normalized IOR-OS-14, IOR-OS-18, MG-63, SAOS-2, U-2OS), and with the MAS5.0 algorithm within the GCOS program of Affymetrix rhabdomyosarcoma (A204, RD, RH-18, RH-30, RH-41, RMS-1, (Affymetrix, Inc.). Samples of pediatric malignancies and cell lines RMS-YM) (17). In addition, IGR-N91 neuroblastoma, SF188 high- were compared to those of adult cancer and normal tissues within the grade glioma, and RES186 pilocytic astrocytoma cell lines were used. R2 database. SUP-B15 was maintained in McCoy's 5A medium containing 20% fetal calf serum (FCS); SF188 and RES1 in Dulbecco‘s minimum Experimental in vivo design. Experiments were carried out under the essential medium Glutamax containing 10% FCS; all other cell lines conditions established by the European Community (Directive were cultured in RPMI-1640 containing 10% FCS at 37˚C and 5% 86/609/CCE). Antitumor activity was evaluated against advanced- CO (all Life Technologies, Saint Aubin, France). Cells were tested to 2 stage subcutaneous RMS-1 rhabdomyosarcoma or SJ-NB-6 be free of mycoplasma. neuroblastoma xenograft tumors in female SPF-Swiss athymic mice as described previously (21). Volasertib was administered Dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) intravenously at 30 and 40 mg/kg once weekly for 4 or 5 weeks; cell-proliferation assay. Cells were seeded at 5,000 or 25,000 per well controls received 0.9% NaCl and 0.1 N HCl vehicle. Statistical in 96-well plates. After 24 hours, volasertib was added to final significance was determined using the two-tailed non-parametric concentrations of 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 and Mann–Whitney or Kruskal–Wallis test and Prism® software version 100000 nmol/l and cells further incubated for 72 h. Cell viability was 3.00 (GraphPad Software, Inc., La Jolla, CA, USA). determined using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS assay; Promega, Mannheim, Germany). Each concentration and time point was tested in quadruplet and all Results experiments in triplicates. Means and standard deviations were calculated from quadruple optical density (OD) measurements. PLK1 mRNA is expressed in pediatric primary malignancies Viability of treated cells was compared to that of untreated cells and to and cell lines. Gene-expression profiling showed PLK1 that prior to exposure, and drug concentrations which inhibited expression in all pediatric primary tumor samples and cell cell growth by 50% (GI50) and reduced viability by 50% (LC50) were calculated as described previously (18). lines tested (Figure 1). Compared to normal tissues, higher Combination studies had been performed using vincristine or levels of PLK1 expression were observed in Ewing sarcomas, etoposide with volasertib at various concentration simultaneously or medulloblastomas, neuroblastomas and rhabdomyosarcomas, not. Combination effects were estimated by combination indices while expression of acute lymphoblastic leukemias and

600 Abbou et al: PLK1 Inhibition in Pediatric Cancer

Figure 1. Gene expression of polo-like kinase 1 (PLK1) in primary tumor tissues and cell lines of pediatric malignancies. PLK1 gene expression using HG-U133 Plus 2.0 microarrays. The dots represent expression levels in individual samples and the gray lines the mean expression level for the respective group of analyzed tissues and cell lines.

osteosarcomas was within the range of normal tissues. All Thus, volasertib exhibits significant antiproliferative activity pediatric tumor cell lines were characterized by high PLK1 at concentrations in the nanomolar range in various pediatric expression. cell lines independently of the tumor type.

Volasertib exhibits significant cytotoxicity against pediatric Volasertib cytotoxicity is mediated by arrest in G2/M and cell lines in vitro. We evaluated volasertib against a large prophase followed by apoptotic cell death. To explore the panel of different pediatric tumor cell lines and cell viability mechanism by which volasertib exhibits its activity, we was reduced in all 40 cell lines tested (Figure 2; Table I). The treated RMS-1 and RD rhabdomyosarcoma, SJ-NB-6, IMR- calculated mean GI50s per cell line ranged from 3.56 nmol/l 32 and IGR-N91 neuroblastoma, SF188 and RES186 glioma to 5045 nmol/l. The RMS-1 and RH-41 alveolar with 10 and 100 nM volasertib and first performed cell- rhabdomyosarcoma, POE Ewing sarcoma, Med-Meb-8A cycle analysis using flow cytometry. In the most sensitive medulloblastoma, IMR-32 neuroblastoma, and HpB-ALL RMS-1 cells (Figure 3A), volasertib resulted in dose- leukemia were most sensitive to volasertib, with GI50s <10 dependent G2/M arrest starting at 6 h and being complete at nmol/l. Thirty-seven out of 40 cell lines tested exhibited GI50 24 h. The effects were less pronounced at 10 nM and cell- values <1210 nmol/l (6/7 Ewing sarcoma, 7/7 leukemia, 5/5 cycle arrest was overcome at 72 hours. However, at 100 nM medulloblastoma, 7/7 neuroblastoma, 5/7 osteosarcoma, and volasertib arrested 72% cells in G2/M which remained 7/7 rhabdomyosarcoma) which corresponds to the maximum persistent up to 72 h. Of note, 14% of cells were found in plasma concentration of volasertib at the recommended dose >4N at 24 h. in the adult phase I study (300 mg) (22). In contrast in neuroblastoma cells, G2/M arrest was The LC50s determined for volasertib on the cell line panel incomplete for IMR-32 (Figure 3B) and SJ-NB-6, and the ranged from 7.9 nmol/l to 24200 nmol/l with overall a mean cell-cycle distribution was returned to baseline at 72 h even LC50 of 2750 nmol/l and a median LC50 of 47.5 nmol/l. RMS- in response to 100 nM volasertib, a dose above the GI50s in 1 rhabdomyosarcoma, Med-Meb-8A medulloblastoma, and these cells. HpB-ALL leukemia were most sensitive to volasertib with In the least sensitive RES186 low-grade glioma (GI50 LC50s <10 nmol/l. LC50s below 1240 nmol/l were observed >100 nM) (Figure 3C), we observed an accumulation of for 27 out of 40 cell lines (5/7 Ewing sarcoma, 7/7 leukemia, tetraploid (4N) cells at 24 h to 100 nM volasertib. At 72 h, it 4/5 medulloblastoma, 7/7 neuroblastoma, 3/7 rhabdomyo- was impossible to distinguish further the different cell sarcoma, and 1/7 osteosarcomas). populations, however, a few >4N cells remained.

601 ANTICANCER RESEARCH 36: 599-610 (2016)

Figure 2. In vitro sensitivity to volasertib in the Innovative Therapies for Children with Cancer core cell line panel. 50% Growth-inhibitory concentration (GI50) (A) and 50% lethal concentration (LC50) (B) are presented for each cell line. The mean GI50 and LC50 for the drug were calculated from all experiments (lower line). The upper line represents 1210 nM which was the maximum concentration of volasertib in adult patients with cancer determined in the clinical phase I study (24).

Using video imaging on unsynchronized cells in order to RMS-1 rhabdomyosarcoma and SJ-NB-6 neuroblastoma that follow cell mitosis under treatment, we found that volasertib- exhibited GI50s of 5 and 20 nM respectively (Table II, Figure treated SF188 cells stopped in prophase and detached from 4). Animals bearing RMS-1 tumors of 74 to 266 mm3 were plates whereas control cells proceeded through mitosis (Figure randomly assigned to treatment groups 19 days after xenograft 3D). Further western blot analysis for key regulators of transplantation. Volasertib administrations at 30 and 40 mg/kg apoptotic cell death showed PARP1 cleavage in both sensitive once weekly were chosen based on the tolerance and activity SF188 and RMS-1 cell lines at 24 and 48 hours, while caspase in prior experiments performed at Boehringer Ingelheim (12). 3 cleavage was detected in SF188 cells at 24 hours and less in Animals of both treatment groups gained body weight and no RMS-1 (Figure 3E). toxicity was noted. RMS-1 xenografts were highly sensitive Thus, complete and persistent G2/M arrest in prophase to volasertib in a dose-dependent fashion. Volasertib at followed by apoptotic cell death was associated with cytotoxic 30 mg/kg resulted in one partial and two complete responses activity of volasertib. Overcoming the G2/M checkpoint and out of nine tumors and significant median tumor growth delay >4N cells may represent an escape mechanism from PLK1- of 36 days (p<0.001; Kruskal–Wallis), 40 mg/kg resulted in inhibiting treatment. 100% tumor regression with two partial and seven complete responses out of nine tumors and a median tumor growth Antitumor activity in vivo of volasertib against RMS-1 and SJ- delay of 39 days (p<0.001). In contrast, SJ-NB-6 NB-6 xenografts. We then evaluated volasertib in vivo against neuroblastoma tumors of 73 to 345 mm3 treated from 21 days

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Table I. 50% Growth-inhibitory concentration (GI50) and 50% lethal concentration (LC50) of volasertib in Innovative Therapies for Children with Cancer cell lines after 72 h exposure determined by dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium assay.

Cell line GI50 (nmol/l) LC50 (nmol/l)

Mean±SD Mean±SD Mean±SD Mean±SD

Ewing sarcoma A673 28.1±0.91 742±2,279 70.3±24.0 3,850±8,907 EW 7 28.0±3.64 47.3±0.43 ORS 5,045±394 24,221±7,587 POE 5.48±2.31 12.4±5.94 RD-ES 17.5±9.01 40.5±6.60 SIM(EW27) 44.5±2.52 2,515±1,395 STA-ET-1 29.1±0.23 47.7±1.69 Leukemia HpB-ALL 4.15±1.56 22.9±16.0 8.08±1.96 39.7±23.0 KOPN-8 37.9±6.11 83.3±3.85 MOLT-16 33.2±2.89 42.8±3.21 REH 12.0±12.9 33.2±21.3 SEM 15.3±7.88 30.9±15.1 SUP-B15 27.1±5.12 38.6±5.32 UOC-B6 31.0±0.84 41.3±1.40 Medulloblastoma DAOY 18.8±12.2 129±228 32.7±20.1 1,268±2,633 D283 MED 29.0±2.14 47.8±0.56 D341MED 29.7±3.29 46.8±1.23 MED-MEB-8A 4.47±0.31 7.86±1.43 UW228.2 561±117 6,207±1,670 Neuroblastoma IMR-32 5.02±3.37 19.5±10.8 21.6±22.5 54.7±44.7 NGP 22.4±3.18 40.1±1.18 SH-SY5Y 24.7±8.60 44.9±5.51 SJ-NB-6 13.1±11.1 100±109 SJ-NB-8 10.3±1.43 43.1±2.86 SK-N-AS 29.7±2.94 46.3±0.82 SK-N-BE 31.2±4.63 86.8±18.8 Osteosarcoma HOS 11.9±2.09 802±1,708 119±94.6 8,530±5,161 IOR-OS-9 34.6±5.57 9,137±492 IOR-OS-14 43.8±4.59 10,984±4,045 IOR-OS-18 4,228±1,713 8,339±53 MG-63 51.1±9.21 10,463±2,812 SAOS-2 30.1±17.2 6,811±1,678 U-2OS 1,214±1,985 13,854±8,406 Rhabdomyosarcoma A204 55.7±32.8 111±173 2,988±1,545 2,355±2,364 RD 26.1±1.49 50.2±11.1 RH-18 219±229 6,158±1,174 RH-30 21.7±17.8 31.0±20.6 RH-41 3.70±0.41 3,876±111 RMS-1 3.56±0.36 9.40±0.29 RMS-YM 444±11.4 3,370±697

after transplantation showed no tumor regression or growth interfering in the cell cycle. The concentration for inhibition delay to volasertib. of 50% of proliferation was determined for each drug alone as Thus, volasertib exhibited a significant antitumor activity 12 nM, 0.5 nM and 500 nM respectively for volasertib, against rhabdomyosarcoma xenografts. vincrinstine and etoposide. According to the Chou and Talalay method, RMS-1 cells were treated with volasertib (at 6, 9, Cytotoxicity of volasertib in combination with cytotoxic 13.5, 20.25 nM) and vincristine (at 0.26, 0.4, 0.5 nM) or agents. In order to determine the potential therapeutic use of etoposide (at 10, 50, 500 nM) which are fractions or multiples volasertib in clinical pediatric protocols, we performed of the GI50 concentration for each drug. combination studies on RMS-1 cells with cytotoxic agents, an Vincristine and volasertib administered simultaneously inhibitor of topoisomerase II and a , both also showed synergistic cytotoxicity, with a CI of <0.9 at fractions

603 ANTICANCER RESEARCH 36: 599-610 (2016)

Figure 3. Continued.

604 Abbou et al: PLK1 Inhibition in Pediatric Cancer

Figure 3. Volasertib induces G2/M arrest in prophase and apoptotic cell death. A, B and C: Cell-cycle analyses showing the number of cells plotted against DNA content. Diploid cells are at 200 and tetraploid cells (G2/M cells) at 400. Sub-G1 cells are represented by <200 fraction and hyperploids cells (>4N) by >400. D: Video imaging of SF 188 cells incubated with volasertib or control. Cell DNA is stained with SYTO® 59 (red) and cytoplasm with CFSE (green). Arrows indicate cell mitosis resulting in two different cells in controls and cell arrest in prophase in response to volasertib treatment. E: Immunoblotting for caspase 3 and poly [ADP-ribose] polymerase 1 (PARP1) cleavage of RMS-1 and SF 188 cells treated with or without volasertib. DMSO: Dimethyl sulfoxide.

of ca. 0.7 and 1.0 of the GI50s (Figure 5A and B). Cell viability by volasertib alone was always higher than the combinations was significantly reduced with the combination at 9 nM at 13.5 nM (p<0.001) and 20.25 nM of volasertib (p<0.0001). (p<0.0001) and 13.5 nM of volasertib (p<0.05). Conversely, Since cell sensitivity to these agents may depend on the cell the combination of volasertib and etoposide was antagonistic, cycle, we further performed combination cell viability tests with CIs of >1 (Figure 5C and 5D). Inhibition of cell viability with different scheduling of both agents. A delay of 24 h

605 ANTICANCER RESEARCH 36: 599-610 (2016)

Figure 4. Antitumor activity of volasertib in RMS-1 rhabdomyosarcoma and SJ-NB-6 neuroblastoma xenografts. Groups of five to seven animals bearing RMS-1 (A) or SJ-NB-6 tumors (B) were treated with volasertib administered intravenously once weekly at 30 or 40 mg/kg for 4 or 5 weeks. Graphs show the mean of each treatment group; bars represent the standard error of mean.

Table II. Antitumor activity of volasertib against RMS-1 and SJ-NB-6 xenografts in mice.

Treatment Total dose, Tumor, n Mean tumor volume DT, days PR CR 5×Vi,days TGD, days p-Valuea mg/kg (range), mm3

RMS-1 Control 7 176 (88-266) 4.8 22.1 Volasertib 30 mg/kg×5 w 150 9 156 (80-242) 1 2 58.4 36.3 <0.001 Volasertib 40 mg/kg×4 w 160 9 145 (74-246) 2 7 61.4 39.3 <0.001 SJ-NB-6 Control 8 155 (92-345) 6.4 17.1 Volasertib 40 mg/kg×4 w 160 8 160 (79-310) 0 0 15 –2.1 ns

DT: Tumor doubling time; PR: partial regression; CR: complete regression; 5×Vi: 5 times initial tumor volume; TGD: tumor growth delay; amedian 5 × initial tumor volumes in treatment groups were compared using a two-tailed non-parametric Kruskal–Wallis test.

between drug addition in medium was chosen based on our with etoposide was strongly antagonistic and reduced the observation that cell-cycle arrest in G2/M phase of RMS-1 effects of volasertib. cells was effective after 24 h of volasertib exposure. For scheduling combination test with volasertib and Discussion vincristine, lower concentrations of 6 and 0.26 nM, respectively, were chosen based on the high cytotoxicity of the simultaneous This study shows the preclinical activity of the newly developed PLK1 inhibitor volasertib in a wide panel of pediatric combination at GI50s of both agents. Adding vincristine before or after volasertib (Figure 4E) resulted in decreased cytotoxicity malignancies independently of tumor histology. We confirmed as compared to simultaneous administration of both agents. In that complete and prolonged G2/M arrest in prophase and contrast, the combination of etoposide added 24 hours after subsequent induction of apoptotic cell death is also the main volasertib was more efficient in cell cytotoxicity than the reverse mechanism of action for the cytotoxicity of this class of agents or simultaneous combination of both agents (Figure 4F). towards pediatric cell lines. Moreover, we demonstrated that Nevertheless, all etoposide combinations significantly abrogated synergistic activity is achieved with spindle cell mitotic poisons the single-agent activity of volasertib alone. when combined simultaneously with PLK1 inhibition. Thus, synergistic cytotoxicity of volasertib and vincristine In the large R2 database, PLK1 mRNA expression in was observed at submaximal concentrations of both agents and pediatric tumor samples was comparable to that in tumors from when administered simultaneously, whereas the combination adult and higher expression levels were observed in pediatric

606 Abbou et al: PLK1 Inhibition in Pediatric Cancer

Figure 5. Volasertib combination with vincristine and etoposide. The percentage cell viability of RMS-1 cells treated with volasertib in combination with (A) vincristine (VCR) or (C) etoposide (VP16) at 72 h is shown. Combination index (CI) calculated by the Chou and Talalay method shows synergism (<0.9) for volasertib and vincristine at concentrations around the 50% growth-inhibitory concentration (B) but antagonism (>1.1) with etoposide (D). Scheduled combination cell-viability assays show that addition of vincristine 24 h before or after volasertib reduces the combination effects as compared to simultaneous administration (E). Adding etoposide 24 h after volasertib leads to more efficient cytotoxicity than the reverse scheduling but all combinations result in reduced cytotoxicity as compared to volasertib alone (F). *p<0.05; **p<0.001; ***p<0.0001.

tumor cell lines compared to primary tumor samples. Although dose of 300 mg volasertib given as an intravenous infusion PLK1 expression has been associated with poor outcome in (24). Therefore, we considered most of our cell lines as being some diseases including neuroblastoma (5, 6, 23), expression sensitive to volasertib. Nevertheless, those considered resistant levels have not been strictly linked to the inhibition of PLK1 were mostly found among the osteosarcoma cell lines. These activity. In this regard, ALL and osteosarcoma cell lines were results were consistent with in vitro activity of volasertib in found to have the lowest PLK1 expression levels compared to previous pediatric studies (16, 25). the other pediatric tumor types which were not reflected by In our mechanistic studies, we showed that complete, lower sensitivity in vitro in the ALL samples. Overall within prolonged G2/M arrest in prophase followed by apoptotic the 40 cell lines tested, GI50s and LC50s ranged over one log- cell death (sub-G1 cells) is the main mechanism of scale. The phase I trial in adult patients found a maximum cytotoxicity to volasertib. Our findings are consistent with plasma concentration of 1210 nM at the recommended phase II the data in adult cell lines with volasertib (12) and the

607 ANTICANCER RESEARCH 36: 599-610 (2016) preceding PLK1 inhibitor BI 2536 (25, 26), suggesting that synchronizes cells in , whereas stabilizes PLK1 depletion or inhibition leads to a poor formation of the mitotic spindle in metaphase, both events that occur after bipolar spindle (4) and apoptosis (15, 27). We found a dose- the effects in prophase mediated by volasertib. Our results dependent effect, and overoming G2/M arrest as observed in highlight the importance of exploring various scheduling for neuroblastoma cells seemed to be one mechanism of escape. drug combinations with anti-mitotics and mechanistic studies A second mechanism of escape appeared to be an before using new association in clinical study. accumulation of tetraploid and >4N cells. This was noted Finally, in vivo experiments showed high sensitivity of only in a limited number of cells in two different sensitive RMS-1 alveolar rhabdomyosarcoma, with 100% tumor cell lines but reflected the main cell DNA distribution pattern responses to 40 mg/kg volasertib, whereas SJ-NB-6 in the resistant RES186 low-grade glioma cell line. Cells neuroblastoma failed to respond to volasertib despite similar began a new cycle without ending mitosis. Hyperploidy had in vitro sensitivity. Recently a novel, distinct mode of action been described as being induced by mitotic spindle poison for PLK1 was discovered (30). PLK1 is a kinase involved in as an escape mechanism (28). the phosphorylation and activation of paired box 3-forkhead Since some cells are able to overcome this prometaphase box O1 (PAX3–FOXO1), the hallmark of alveolar arrest, it appears intriguing to add other spindle assembly rhabdomyosarcoma, and required for expression of several inhibitors. We found that treatment of volasertib combined with direct transcriptional targets (31). This additional role of PLK1 the vincristine had a strong synergistic effect on in PAX3–FOXO1 fusion-positive rhabdomyosarcoma may cytotoxicity. As shown for the RMS-1 rhabdomyosarcoma cell underly the sensitivity observed in RMS-1 cells and our line, synergy was observed on one hand at 'sub-maximal' findings in RMS-1 tumors further support this new hypothesis. concentrations of both agents, i.e. doses around the GI50, and PLK1 inhibition using volasertib represents a promising on the other hand when drugs were given simultaneously rather novel therapeutic for pediatric cancer. Its cytotoxic activity than successively with time delay. Our data confirm and extend against a wide range of tumor types mediated by pro- those reported by the group of Fulda in neuroblastoma cell metaphase arrest and induction of apoptosis, synergistic activity lines with the previous PLK1 inhibitor BI 2536 (15) and in with other mitotic spindle poisons, and the particular sensitivity rhabdomyosarcoma cells with volasertib (27). Combination in vivo of PAX3–FOXO1 fusion-positive rhabdomyosarcoma with vinca induced apoptosis in vitro and suppressed motivate further clinical evaluation of volasertib in combination tumor growth in vivo. The main underlying mechanisms of the with other agents. Our results further highlight the major synergism of the combination was found to be the mitotic importance of the current preclinical combination study for arrest and activation of both caspase-dependent and caspase- evaluation of new therapeutics. independent apoptosis through down-regulation of induced myeloid leukemia cell 1 (MCL1), B-cell lymphoma 2 (BCL2) Acknowledgements and B-cell lymphoma-extra large (BCL-XL) inactivation, BCL2-associated X (BAX)/BCL2 homologous The Authors are grateful to the Platform of Preclinical Evaluation antagonist/killer (BAK) activation and reactive oxygen species (PFEP) and Dr Patrick Gonin for providing immunocompromised production (27). Given the main activity of volasertib during mice and health care of animals, and Sophie Salome-Desnoulez for prophase and the mechanisms of vinca alkaloid as mitotic assistance of the video imaging. They, also, thank Carole Lecinse for critical reading of the manuscript. spindle poison, we have shown the importance of simultaneous administration to maximize the combined effects. In contrast, the combination of volasertib with etoposide References was highly antagonistic and resulted in abrogation of cytotoxic effects of volasertib. Etoposide inhibits 1 Mundt KE, Golsteyn RM, Lane HA and Nigg EA: On the topoisomerase II and induces G /M arrest before mitotic regulation and function of human polo-like kinase 1 (PLK1): 2 effects of overexpression on cell-cycle progression. Biochem entry (29). As the main mechanism of cytotoxicity of Biophys Res Commun 239: 377-385, 1997. volasertib is a pro-metaphase arrest, etoposide-mediated cell- 2 Roshak AK, Capper EA, Imburgia C, Fornwald J, Scott G and cycle arrest before mitotic entry could by-pass the action of Marshall LA: The human polo-like kinase, PLK, regulates volasertib on mitosis and abrogate or delay its cytotoxicity. CDC2/ through phosphorylation and activation of the This hypothesis is supported by our findings that CDC25C phosphatase. Cell Signal 12: 405-411, 2000. administration in vitro of etoposide after volasertib resulted 3 Inoue D and Sagata N: The Polo-like kinase Plx1 interacts with in less antagonistic effects on volasertib cytotoxicity than the and inhibits Myt1 after fertilization of Xenopus eggs. EMBO J 24: 1057-1067, 2005. reverse or simultaneous administration. Drug scheduling 4 Casenghi M, Meraldi P, Weinhart U, Duncan PI, Körner R and might also be important for the combination with Nigg EA: Polo-like kinase 1 regulates Nlp, a centrosome protein and paclitaxel, which failed to show synergistic involved in microtubule nucleation. Developmental Cell 5: 113- effects in rhabdomyosarcoma cells (27). Doxorubicin 125, 2003.

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Pagès, Gilles Vassal, Dieter Zopf and Birgit Geoerger: Regorafenib: antitumor activity upon mono and combination Received December 1, 2015 therapy in preclinical pediatric malignancy models. Plos One Revised January 15, 2016 10(11): e0142612, 2015. Accepted January 20, 2016

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