AuroraAKinaseInhibitionDestabilizesPAX3-FOXO1and MYCN and … · tech) on plates coated with Matrigel (Corning Life Sciences). In vitro drugs screenings Cells were seeded in 384-well

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Aurora A Kinase Inhibition Destabilizes PAX3-FOXO1 andMYCN and Synergizes with Navitoclax to InduceRhabdomyosarcoma Cell Death A C

Johannes Ommer1, Joanna L. Selfe2, Marco Wachtel1, Eleanor M. O'Brien2, Dominik Laubscher1,Michaela Roemmele1, Stephanie Kasper1, Olivier Delattre3, Didier Surdez3, Gemma Petts4, Anna Kelsey4,Janet Shipley2, and Beat W. Sch€afer1

ABSTRACT◥

The clinically aggressive alveolar rhabdomyosarcoma (RMS)subtype is characterized by expression of the oncogenic fusionprotein PAX3-FOXO1, which is critical for tumorigenesis andcell survival. Here, we studied the mechanism of cell deathinduced by loss of PAX3-FOXO1 expression and identified anovel pharmacologic combination therapy that interferes withPAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsicapoptosis in a NOXA-dependent manner. This was pharmaco-logically mimicked by the BH3 mimetic navitoclax, identified astop compound in a screen from 208 targeted compounds. In aparallel approach, and to identify drugs that alter the stability ofPAX3-FOXO1 protein, the same drug library was screened andfusion protein levels were directly measured as a read-out. Thisrevealed that inhibition of Aurora kinase A most efficiently

negatively affected PAX3-FOXO1 protein levels. Interestingly,this occurred through a novel specific phosphorylation event inand binding to the fusion protein. Aurora kinase A inhibitionalso destabilized MYCN, which is both a functionally importantoncogene and transcriptional target of PAX3-FOXO1. Com-bined treatment with an Aurora kinase A inhibitor and navi-toclax in FP-RMS cell lines and patient-derived xenograftssynergistically induced cell death and significantly slowed tumorgrowth. These studies identify a novel functional interaction ofAurora kinase A with both PAX3-FOXO1 and its effectorMYCN, and reveal new opportunities for targeted combinationtreatment of FP-RMS.

Significance: These findings show that Aurora kinase A andBcl-2 family proteins are potential targets for FP-RMS.

IntroductionRhabdomyosarcoma (RMS) are the most common pediatric soft-

tissue sarcoma. The most aggressive subtype, alveolar RMS, is char-acterized by the occurrence of balanced reciprocal translocations,resulting in expression of oncogenic fusion proteins (1, 2), wherebythe most common fusion is PAX3-FOXO1. Fusion-positive RMS (FP-RMS) has a high propensity tometastasize, and resistance to standard-of-care treatments is common, resulting in 5-year survival rates of onlyabout 30% (3–5). The search for novel targeting strategies is difficult aspediatric tumors generally harbor only few somatic mutations (6),which is especially true for the FP-RMS subtype (7). The lack of othersomatic mutations underscores the important role of PAX3-FOXO1,which functions as transcriptional activator affecting multiple onco-genic pathways (8). We previously demonstrated that antisense-

mediated loss of PAX3-FOXO1 results in cell death, underscoringthe addiction of FP-RMS cells to the fusion protein.However, so far theexact mechanism by which the cells die has not been described (9).

Due to its importance for tumor cell survival, therapeutic targetingof PAX3-FOXO1 has become a paramount goal of RMS research. Oneapproach has been to identify key downstream effectors of the fusionprotein as potential therapeutic targets, such asMYCN (10). However,because MYCN and PAX3-FOXO1 are transcription factors lackingenzymatic activity, targeting these proteins directly is challenging.Recently, new strategies have been developed targeting PAX3-FOXO1indirectly by inhibiting its stabilizer Polo-like kinase 1 (PLK1; ref. 11)or its cofactors, like bromodomain-containing protein 4 (BRD4;ref. 12) or chromodomain-helicase DNA-binding protein 4 (CHD4;ref. 13). Further strategies to target PAX3-FOXO1 and MYCN arereviewed in refs. 14, 15. Although single-target therapies can beefficient in preclinical models and clinical treatment, they are proneto develop therapy resistance. One way to circumvent this problem isto use drug combinations that target different pathways preventingescape.

Recently, we showed that PLK1 stabilizes PAX3-FOXO1 throughphosphorylation of serines 503 and 505, protecting the fusion proteinfrom proteasomal degradation (11). Although single-agent inhibitionof PLK1 proved very effective in reducing tumor growth in vivo,resistance against this treatment was also observed. One of theregulators of PLK1 is Aurora A kinase (AURKA), which thereforemight also functionally contribute to FP-RMS tumorigenesis. Aurorakinases are a family of serine-threonine kinases that are involved incell-cycle progression, most importantly during mitosis (16). AURKAis highest expressed at the G2–M transition (17) where it activatesPLK1 by phosphorylation at threonine 210 as crucial step for check-point recovery (18, 19). Furthermore, AURKA serves important roles

1Department of Oncology and Children's Research Center, University Children'sHospital Zurich, Zurich, Switzerland. 2Sarcoma Molecular Pathology Laboratory,The Institute of Cancer Research, London, United Kingdom. 3France INSERM

U830, �Equipe Labellis�e LNCC, PSL Universit�e, SIREDO Oncology Centre, InstitutCurie, Paris, France. 4Department of Diagnostic Paediatric Histopathology,Royal Manchester Children's Hospital, Manchester, United Kingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Beat W. Sch€afer, Children's Hospital Zurich, Steinwies-strasse 75, 8032 Zurich, Switzerland. Phone: 41-44-266-7553; Fax: 41-44-266-7171; E-mail: [email protected]

Cancer Res 2020;80:832–42

doi: 10.1158/0008-5472.CAN-19-1479

�2019 American Association for Cancer Research.

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in centrosomematuration andmitotic entry (20), as well as for spindleassembly (21). These functions in cell-cycle progression indicate animportant role for Aurora kinases in cancer. Indeed, AURKA isupregulated in a variety of tumors and has thus become a focus ofinhibitor development (22–25). AURKA has also been shown tophosphorylate AKT and mTOR, indicating a role in promotingchemotherapy resistance (26).

Our goal was thus to find a novel synergistic combination therapy totreat FP-RMS. We demonstrate that FP-RMS cells undergo intrinsicapoptosis in a NOXA-dependent manner after loss of PAX3-FOXO1,which can be enhanced by the BH3-mimetic navitoclax (ABT-263).Moreover, we established a novel functional link betweenAURKA andstability of both PAX3-FOXO1 and MYCN. Combination of navito-clax and alisertib had synergistic antitumor effects in vitro and in vivoand thus provides the basis for a promising new rationale combinationtherapeutic approach for FP-RMS.

Materials and MethodsCell lines

RMS cell lines RD, Rh4 (Peter Houghton, St. Jude Children'sHospital, Memphis, TN), RhJT (Scott Diede, Fred Hutchinson CancerResearch Center, Seattle, WA), RMS (Janet Shipley, Sarcoma Molec-ular Pathology, The Institute of Cancer Research, London, UK), KFR(Jindrich Cinatl, Frankfurter Stiftung f€ur krebskranke Kinder, Frank-furt, Germany), Rh30 as well as HEK293T cells (both ATCC LGCPromochem) were cultured in high-glucose DMEM (Sigma-Aldrich),supplemented with 100 U/mL penicillin/streptomycin, 2 mmol/LL-glutamine, and 10% FBS (Life Technologies) at 37�C and 5% CO2.All RMS cell lines were authenticated upon receipt by short tandemrepeat (STR) profiling and used for experimentation from frozenstocks within 10 to 20 further passages. As the human myoblast cellline has not yet been characterized by STR profiling, negativematchingwith all available cell lines in the database was used for verification. Allcells were regularly tested for mycoplasma contamination by aPCR-based assay.

Cells from patient-derived xenograftsPatient-derived xenograft (PDX) tumors were dissociated as

described before (27). In brief, tumor tissue was minced withscalpels and suspended in Hanks' Balanced Salt Solution (Sigma-Aldrich) supplemented with 1 mmol/L MgCl2, 200 mg/mL Lib-erase, and 200 U/mL DNase I (both Roche). Tissue was digestedfor 30 minutes at 37�C and filtered twice through 70 mm cellstrainers (BD Biosciences). Dissociated cells were washed withPBS (Sigma-Aldrich) before freezing or resuspending for furtherculture.

Cells derived from PDX tumors were cultured in Neurobasalmedium (Life Technologies) supplemented with 2x B-27 Supplement(Life Technologies), 20 ng/mL EGF, and 20 ng/mL basic FGF (Pepro-tech) on plates coated with Matrigel (Corning Life Sciences).

In vitro drugs screeningsCells were seeded in 384-well plates, and shRNA was induced by

100 ng/mL doxycycline (Sigma-Aldrich). Drugs (SupplementaryTable S1) were purchased from Selleckchem. Twenty-four hoursafter seeding cells, medium was changed to 19 mL culture medium.Drugs were prediluted to 10 mmol/L in culture medium, and 1 mLof each drug was added to the wells for a final concentration of500 nmol/L. Viability was measured after 48 hours by WST-1assay.

Cells for immunoblot analysis were seeded in 24-well plates anddrugs added at 100 nmol/L after 48 hours of incubation.

Mouse xenograft experimentsNOD/Scid il2rg�/� (NSG) mice were 8 to 12 weeks old for the

experiments. Note that 5� 106 Rh4 or IC-PPDX-35 cells were injecteds.c. into the flanks. After engraftment mice were randomized into fourgroups (5–6 mice per group) when tumors reached 100 mm3. Tumorgrowth was assessed by caliper measurements, and the volumes werecalculated using the formulaV¼ (4/3) p r3; r¼ (d1þd2)/4. Mice weresacrificed when the tumor volume reached 1000 mm3. All animalexperiments have been approved by the Swiss veterinary authorities(license ZH206/15).

Statistical analysisData analysis was performed with GraphPad Prism 7. Significance

was calculated using unpaired two-tailed Student t test or Welch two-tailed test. Two-way ANOVA was used for multiple comparisons.Differences were considered statistically significant withP< 0.05. Drugsynergy was calculated using the Bliss independence model in the freeSynergyFinder WebApp (28).

For additional methods, see SupplementaryMaterials andMethodsSection.

ResultsSilencing of PAX3-FOXO1 expression induces intrinsicapoptosis in a NOXA-dependent manner

Because it was previously shown that silencing of PAX3-FOXO1expression results in FP-RMS cell death (9), we aimed first to furtherelucidate its precise mechanism.

We generated FP-RMS cell lines expressing an inducible shRNA(shP3F) or control (shsc) to specifically silence PAX3-FOXO1 expres-sion upon doxycycline treatment (Supplementary Fig. S1A). Afterconfirming that the system significantly downregulated the fusionprotein both at the mRNA and protein levels in four different cell lines(Supplementary Fig. S1B–S1D), we assessed whether cells wouldundergo apoptosis. We observed a significant increase in caspase-3/7 activity in Rh4 and Rh30 cells after PAX3-FOXO1 depletioncompared with control (Fig. 1A; Supplementary Fig. S1E). Thisincrease was also reflected by the appearance of cleaved products ofPARP, caspases 3, 7, and 9 (Fig. 1B). To exclude off-target effects, weoverexpressed a nontargetable PAX3-FOXO1 mutant, which indeedcould rescue cells from apoptosis despite silencing of the endogenousfusion protein as shown by reduced caspase-3/7 activity (Supplemen-tary Fig. S1F) and decreased levels of cleaved PARP and caspase-3protein products (Supplementary Fig. S1G).

To further confirm this notion, we also treated cells after fusionprotein depletion with increasing concentrations of the pan-caspaseinhibitor zvad-FMK, which again could restore viability of shP3F-Rh4 cells (Fig. 1C) and reduced cleaved PARP and caspase-3protein levels (Supplementary Fig. S1H). To exclude other modesof cell death, we also treated cells with the different cell deathinhibitors zvad-FMK (apoptosis), necrostatin-1 (necroptosis), fer-rostatin (ferroptosis), as well as E64d (cathepsin inhibitor) andcathepsin GI. However, only zvad-FMK could rescue viability butnone of the other inhibitors (Fig. 1D). These data indicate thatapoptosis is the major mode of cell death activated in FP-RMS cellsupon depletion of PAX3-FOXO1.

Next, we sought to identify the proapoptotic protein(s) responsiblefor initiating apoptotic cell death. We performed a small-scale

AURKAi and BCL-XLi Synergize to Control FP-RMS Tumor Growth

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CRISPR/Cas9 screen in shP3F-Rh4 cells using the construct depictedin Supplementary Fig. S2A to knock out proapoptotic genes eitherindividually or in combination, and measured cell viability uponPAX3-FOXO1 depletion. Knockdown efficiencies of Bax, Bak, Bad,and Bim as examples are shown in Supplementary Fig. S2B and S2C. Incontrol cells (shsc-Rh4), only depletion of caspase-9, but not caspase-8,and the combination of Bax/Bak were able to significantly reducecaspase-3/7 activity (Supplementary Fig. S2D). This indicates thatactivation of the extrinsic pathway is less important in FP-RMS cells.Upon depletion of PAX3-FOXO1, NOXA was the only BH3-onlyprotein capable to significantly reduce caspase-3/7 activity (Fig. 1E;Supplementary Fig. S2E), similar to knockout of the pore-formingproteins BAX and BAK. These results were validated in threeadditional FP-RMS cell lines (Supplementary Fig. S2F), albeit the

extent of repression differed among cell lines. Nevertheless, NOXAseems to play an important role in initiating apoptosis after PAX3-FOXO1 depletion, although the contribution of other BH3-onlyproteins cannot be excluded. Furthermore, cell-cycle analysis afterPAX3-FOXO1 silencing (shP3FDox) indicated no significantchange in cell-cycle distribution, but appearance of a sub-G1 peakin all cells except Rh30, indicative of induction of apoptosis(Supplementary Fig. S2G). This is in line with the observation thatboth NOXA mRNA and protein expression are upregulated uponsilencing of the fusion protein in all cell lines tested with theexception of Rh30 cells (Fig. 1F and G; SupplementaryFig. S2H). Taken together, our results demonstrate that intrinsicapoptosis initiated upon silencing of PAX3-FOXO1 depends onupregulation of the BH3-only protein NOXA.

Figure 1.

Silencing of PAX3-FOXO1 induces apoptosis via NOXA. A, Caspase activity after silencing of PAX3-FOXO1. Caspase-3/7 activity was assessed 24, 48, and 72 hoursafter induction of shRNA expression in Rh4 cells. Mean of three independent experiments; bars, SD; two-way ANOVA, ��, P � 0.001. B, Western blot analysis ofwhole-cell lysates fromRh4 shsc or shP3F 48 hours after shRNA inductionwith doxycycline (þ) or no induction (�).C,Z-vadmediated rescue from cell death.WST-1assay of Rh4 shsc or shP3F treated with doxycycline and increasing concentrations of z-vad FMK. Mean of two independent experiments; bars, SD; Student t test,� ,P�0.05; �� ,P�0.01.D,Rescue experiment after shRNA-mediated silencing of PAX3-FOXO1mRNA. Rh4 cells expressing either scrambled shRNA (shsc) or shRNAtargeting PAX3-FOXO1 mRNA (shP3F) were treated for 48 hours with 0.1 mg/mL doxycycline to induce shRNA expression. Viability was assessed using WST-1assay and is shown relative to nontreated cells. Several cell death inhibitors were used at the concentration indicated. Mean of two independent experiments; bars,SD. E, BH-3-only protein screen. Caspase-3/7 activity of Rh4 shP3F cells harboring CRISPR/Cas9-induced knockouts of the indicated genes. sc, scrambled sgRNA.White bars, no shRNA induction; black bars, 48 hours after doxycycline-induced shRNA expression. Mean of two independent experiments; bars, SD. F, NOXAexpression after PAX3-FOXO1 silencing. Western blot analysis of whole-cell lysates from Rh4 shsc or shP3F 48 hours after shRNA induction with doxycycline (þ) orno induction (�).G,RelativemRNAexpression ofNOXA inRh4 shsc or shP3F48 hours after induction of shRNAwith doxycycline. Gene expressionwas normalized toGAPDH. Mean of two independent experiments. Each experiment was performed in triplicates.

Ommer et al.

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The BH3-mimetic navitoclax enhances cell death after PAX3-FOXO1 depletion

Next, we aimed to find drugs that would enhance apoptosis inducedby PAX3-FOXO1 depletion. For this, we set up a compound screenof 208 drugs at a final concentration of 500 nmol/L and treatedshP3F-Rh4 cells after doxycycline-mediated shRNA induction (Sup-plementary Fig. S3A; Supplementary Table S1). Results are depicted asratio of viability comparing shP3F versus shsc cells. The screenidentified 13 candidate drugs that decreased viability by at least anadditional 50%, while not affecting control cells (Fig. 2A, left plot; rawdata Supplementary Table S2). Classification of the top hits accordingto their mechanism of action revealed that three of them are BH3-mimetics (Fig. 2A, right plot) with navitoclax (ABT-263) being themost potent drug, also when validating each as single agent (Fig. 2B;Supplementary Fig. S3B–S3M). To directly compare BCL-XL versusBCL-2 targeting, we generated dose–response curves for navitoclaxand venetoclax (ABT-199; Fig. 2C and D). Interestingly, navitoclaxreduced IC50 to at least a 10 times lower concentration (reduction of

3.0 mmol/L to 0.18 mmol/L) than venetoclax (reduction of 9.6 mmol/Lto 5.6 mmol/L). These findings suggest that inhibition of BCL-2 mightbe less important. To confirm this notion, we treated cells withincreasing concentrations of an additional BCL-XL–specific inhibitor,A1331852, andUMI-77, which isMCL-1 specific. AlthoughA1331852showed comparable effects to navitoclax (Fig. 2E), treatment withUMI-77 did not further increase cell death (Supplementary Fig. S3N).These results strengthen the observation that PAX3-FOXO1 silencingprimes FP-RMS cells to apoptosis via balancingNOXAversus BCL-XLlevels.

To demonstrate thismore directly, we treated shP3F-Rh4-NOXA�/�

cells (Supplementary Fig. S2E) with navitoclax and found indeedreduced sensitivity upon silencing of PAX3-FOXO1 (Fig. 2F). Finally,expression analysis of different datasets in the r2 database (R2:Genomics Analysis and Visualization Platform: http://r2.amc.nl)revealed that NOXA expression was elevated at base levels in all RMSdatasets compared with healthy skeletal muscle tissue (SupplementaryFig. S3O).

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Figure 2.

Navitoclax sensitizes cells to cell death after silencing of PAX3-FOXO1.A,Drug screen to enhance cell death after silencing of PAX3-FOXO1. Rh4 shsc and Rh4 shP3Fcells were treated with drugs (Supplementary Table S1) while simultaneously inducing shRNA expression with 0.1 mg/mL doxycycline for 48 hours. Viability wasmeasured by WST-1 assay, and the relative viability effect of each drug was calculated (see Materials and Methods). Left plot, the mean viability from threeindependent experiments performed in duplicates. Right plot, list of the top hits according to the ratio of relative viability of drugs on shP3F over shsc. Rankedaccording to their targeting class.Welch two-tailed t test, � , P� 0.05; �� , P�0.01; �� , P�0.001. B, Individual effect of navitoclax on relative cell viability of Rh4 shscor Rh4 shP3F compared with DMSO. SD; Welch two-tailed t test, �� , P � 0.01. n.s., nonsignificant. C–E, Relative cell viability of Rh4 shsc or Rh4 shP3F cells after48 hours with or without shRNA induction and simultaneous treatment with increasing concentrations of navitoclax, venetoclax, or A-1331852, respectively. Mean�SD from three independent experiments performed in triplicates. IC50 values were calculated from nonlinear regression analysis using GraphPad Prism 7. F, Relativecell viability of Rh4 shsc PMAIP1�/� or Rh4 shP3F PMAIP1�/� cells treated with increasing concentrations of navitoclax for 48 hours.





http://r2.amc.nl


These findings suggest that RMS cells might be already primedtowards a proapoptotic state through higher basal NOXA expres-sion, which can be further enhanced by reduction of fusionprotein levels and renders them more sensitive towards navitoclaxtreatment.

Aurora kinase A inhibition reduces PAX3-FOXO1 proteinstability

Next, we aimed to pharmacologically reduce PAX3-FOXO1 proteinlevels. To this end, we treated wild-type (WT) Rh4 cells with the samedrug library (Supplementary Table S1) and analyzed cell lysates byWestern blot (Supplementary Fig. S4A). To exclude general toxiceffects, all signals were densitometrically digitalized and normalized tothe house keeping proteinGAPDH. Initial hits were calledwhen fusionprotein levels were lowered below 80%, a criterion fulfilled by 43compounds (Fig. 3Aand B; raw data Supplementary Table S3). Whenclassifying these hits according to their drug targets, we identifiedfive epigenetic regulators, though HDAC inhibitors like entinostat,which was recently described to reduce fusion protein levels (29),showed only mild to no effects (Supplementary Fig. S4B). Further-more, we classified five proteasome, five AURKA, and three CDK9inhibitors (Fig. 3C). Strikingly, AURKA inhibitors were alreadyidentified in our apoptosis screen as hits (Fig. 2B), and previouswork from our laboratory demonstrated a direct interactionbetween PAX3-FOXO1 and PLK1, which stabilizes the fusionprotein (11) and is known to be activated by AURKA (19). Hence,we individually validated AURKA inhibitors at increasing doses andidentified alisertib as being the compound active at the lowest dosestested (Fig. 3D; Supplementary Fig. S4C and S4D). Importantly,treatment of PDX-derived primary cells with alisertib also reducedfusion protein levels (Supplementary Fig. S4D). We were theninterested to study the mechanism underlying the functional inter-action of AURKA and PAX3-FOXO1. We treated Rh4 cells withalisertib, which increased total protein levels of both PLK1 andAURKA, whereas we observed a reduction of PLK1 phosphoryla-tion at threonine 210, as expected (Fig. 3E). Because AURKA hasbeen described to phosphorylate S256 in WT FOXO1 (30), we theninvestigated phosphorylation of S437 in PAX3-FOXO1 (corre-sponding to residue S256 in WT FOXO1). Indeed, we also observeda clear reduction at this site upon alisertib treatment (Fig. 3E). Asthis region of PAX3-FOXO1 has been described to be relevant forprotein stability (reviewed in ref. 15), we next studied whetherphosphorylation of S437 contributed to fusion protein stability byreplacing serine with alanine. Interestingly, and actually evenslightly more pronounced than the previously described S503Amutant, the S437A mutant also significantly reduced fusion proteinstability as revealed after 8-hour treatment with cycloheximide(Fig. 3F and G).

Lastly, to investigate a potential direct interaction of AURKA andPAX3-FOXO1, we fused PAX3-FOXO1 to the bacterial biotin-ligaseBirA (31) and expressed it ectopically in HEK293T cells. After pulldown of biotinylated proteins using streptavidin coated beads, PLK1was identified onWestern blots in fusion protein samples but not in theBirA-only control, as shown before (Fig. 3H; ref. 32). Strikingly, alsoAURKA was identified in this assay as being selectively biotinylated,suggesting a novel direct interaction of this kinase and the fusionprotein (Fig. 3H).

Taken together, our results indicate that AURKA inhibition candecrease fusion protein stability through reduced phosphorylation ofserine 437, which might contribute to its ability to induce apoptosis inFP-RMS cells.

Aurora kinaseA inhibition also destabilizesMYCN inFP-RMScelllines

AURKA is reported to stabilize the MYCN protein that is highlyexpressed through transcriptional regulation by the fusion protein andgenomic amplification (10, 33–35). Previous studies showed thatAURKA stabilizes MYCN by interfering with SCFFbxW7-mediatedubiquitination and that sequential phosphorylation of MYC proteinsat S62 and T58 is required for binding of SCFFbxW7 (36, 37). Wetherefore investigated whether AURKA inhibition affected MYCNprotein levels in FP-RMS cells. We stably transduced three differentFP-RMS cell lines with plasmids expressing shRNA against AURKAmRNA. In all cell lines, depletion of AURKA resulted in reducedMYCN protein levels (Fig. 4A). In accordance with published liter-ature (38), MYCN double mutants (T58A/S62A) had more stableMYCN and were rescued from degradation after depletion of AURKAin RMS cells (Fig. 4B). This finding indicates that in FP-RMS cells,MYCN stability is controlled by AURKA through the same mechan-isms described for other cell types and cancers (39). Lastly, we wantedto confirm the influence of pharmacologic AURKA inhibition onMYCN protein levels in FP-RMS cells. We treated different FP-RMScell lines with increasing concentrations of the AURKA inhibitorsalisertib, as well as CCT137690 (a kinase inhibitor), and CD532 (anamphosteric inhibitor with similarities to alisertib), respectively. In allcell lines, inhibition of AURKA resulted in loss of MYCN proteinlevels in a dose-dependent manner at submicromolar doses (Fig. 4C;Supplementary Fig. S5A and S5B).

Taken together, our data show that AURKA stabilizes MYCN inaddition to its effects on PAX3-FOXO1, and its inhibition destabilizesMYCN, a target gene of the fusion protein.

Alisertib and navitoclax act synergistically in vitroBecause it is unlikely that single agents will be able to provide

significant clinical benefit, we next wanted to identify a combination ofsynergistically acting drugs. To do this in an unbiasedway, we screenedour library for compounds that would synergistically reduce cellviability in conjunction with a low concentration of navitoclax(IC20; Supplementary Fig. S6A and S6B; raw data SupplementaryTable S4). We assessed cell viability after 48 hours and ranked theresults according to synergism. Strikingly, out of the 28 hits identified,7 were AURKA inhibitors (Fig. 5A). Six of these were also identifiedin a similar screen carried out in a second FP-RMS cell line (Supple-mentary Fig. S6C and S6D). Hence, AURKA inhibitors might act insynergy with navitoclax. To test this, we treated FP-RMS cells withincreasing concentrations of both alisertib and navitoclax to obtain acombination matrix. Indeed, the combination was able to induce celldeath even at lower drug concentrations (Fig. 5B) and was highlysynergistic as assessed by SynergyFinder (Fig. 5C; SupplementaryFig. S7A; ref. 28). Importantly, we observed comparable synergisticeffects not only in cell lines but also in six PDX-derived primary cells.This synergy was tumor specific as nontumorigenic cells (humanforeskin fibroblasts and myoblasts) were not sensitive toward thecombination treatment despite expression of various levels of NOXA(Supplementary Fig. S7B). NOXA�/� cells were also not sensitive tothe combination treatment, demonstrating thatNOXA expressionwasrequired (Supplementary Fig. S8A and S8B).

As AURKA plays an important role for cell-cycle progressionduring G2–M phase, we assessed cell-cycle distribution after alisertibor combination treatment. Treating cells with 50 or 100 nmol/Lalisertib, we observed an increasing proportion of cells arrested inG2–M phase (Fig. 5D). Combination with 800 nmol/L navitoclaxhowever strongly reduced the G2–M peak and increased the sub-G1

Ommer et al.





Figure 3.

Inhibition of Aurora kinase A leads to reduced PAX3-FOXO1 protein stability.A, PAX3-FOXO1 protein levels were assessed byWestern blot 48 hours after treatmentwith 100 nmol/L of compounds (Supplementary Table S1). Protein bandswere analyzed by densitometry and normalized to GAPDH levels. Treatment was comparedwith DMSO. B, Exemplary blot of one set of drugs. Red, Aurora kinase A inhibitors. C, Chart showing the classes of inhibitors found among the top candidates. Red,Aurora Kinase A inhibitors; green, CDK9 inhibitors; gray, epigenetic modulators. D, Western blot of lysates from Rh4 cells treated for 48 hours with increasingconcentrations of the given drug. Left plot, alisertib; right plot, AT9283. E, Immunoblot for phosphorylation at the indicated sites. Cells were incubated for 24 hourswith alisertib and lysed with coimmunoprecipitation buffer. F,Western blot of RD cells transiently transfected with the respective overexpression plasmid for PAX3-FOXO1 mutants or WT. Eight hours before lysis, cells were treated with either 10 mg/mL cycloheximide (CHX) or DMSO to block protein synthesis. G, Densitometricquantification of the fusion protein levels, normalized to GAPDH. Mean of three independent experiments; bars, SD; two-way ANOVA, � , P < 0.05; �� , P < 0.01.H, Western blot analysis of streptavidin pull-down experiments. HEK293T cells were transduced with a PAX3-FOXO1 expression plasmid fused to BirA biotinligase (P3F-BirA) orwith GFP or BirA alone. Cellswere incubatedwith biotin (þ) or not (�) and lysed. Pull-downwas performedwith beads coatedwith streptavidin.






fraction (Fig. 5D; Supplementary Fig. S8C). This suggests that alisertibalone induces cell-cycle arrest whereas in combination with navitoclaxpushes cells into apoptosis. These findings are also supported by asynergistic increase in caspase-3/7 activity (Supplementary Fig. S8D).Hence, alisertib acts synergistically with navitoclax in vitro to induceapoptosis in cell lines and primary PDX-derived RMS cells, whereasthe drug combination had no major effects on nontumorigenic cells.

Combination of alisertib and navitoclax synergistically reducestumor growth in vivo

Having confirmed a synergistic action of alisertib and navitoclaxin vitro, we next aimed to assess the antitumorigenic response of thecombination in vivo. We injected NSG mice subcutaneously witheither Rh4 or patient-derived IC-PPDX-35 cells. After tumors werepalpable, mice were randomized into four groups and treated dailyover 3 weeks with either vehicle, navitoclax alone (80 mg/kg), alisertibalone (30 mg/kg), or the combination of both drugs (SupplementaryFig. S9A). Although continuous tumor growth was observed in Rh4cells in vehicle and navitoclax-only–treated mice, alisertib treatmentslightly delayed tumor growth but failed to induce lasting effects(Fig. 6A). In contrast, combination therapy resulted in modest tumorregression and lasting stable disease even after end of the treatmentperiod (Fig. 6A). This was also reflected in the survival ofmice, becauseonly animals in the combination group survived (Fig. 6B). In the PDXmodel, alisertib treatment alone provoked a stronger delay in tumorgrowth albeit also in this setting, combination treatment showed themost stable growth control (Fig. 6C) and survival was increased(Fig. 6D). In neither experiment, we observed significant weight loss(Supplementary Fig. S9B and S9C). To investigate whether alisertibtreatment mimics PAX3-FOXO1 depletion, we also performed anin vivo experiment with the doxycycline-inducible shP3F line incombination with navitoclax. Although silencing of the fusion proteinalone already leads to significant growth inhibition, addition ofnavitoclax treatment has a further, albeit small, combinatorial effect(Supplementary Fig. S9D). We also isolated IC-PPDX-35 tumors after1 week of treatment, where histological analysis revealed a marked

increase in the number of apoptotic cells in combination-treatedtumors (Fig. 6E) and increased staining for cleaved caspase-3(Fig. 6E). Consequently, proliferation was reduced as monitored byKi67 staining and increased expression of p21 (Supplementary Fig. S9Eand S9F).

Tounderscore the clinical relevance ofAURKAinhibition,wefinallynoticed that AURKA RNA expression is significantly higher in threeindependent RMS datasets compared with healthy skeletal muscle(Fig. 6F; data from R2 database: http://r2.amc.nl). Further, we dem-onstrated AURKA protein expression in the majority of primary RMS.Twenty-six of 37 (70%) of FP-RMS and 37 of 44 (84%) of FN-RMSstained positively for AURKA by IHC (Supplementary Fig. S10AandS10B). In addition,AURKAand theproliferationmarkerKi-67 showeda significant correlation in FN-RMS and a trend toward significance inFP-RMS (Supplementary Fig. S10C and S10D). This was consistentwith significant correlations between AURKA and MKI67 at themRNA level (Supplementary Fig. S10E and S10F). No significantcorrelations between AURKA IHC scores and event-free or overallsurvival were seen in either the FP or FN groups.

DiscussionOur aim for this study was to identify a novel synergistic combi-

nation treatment strategy based on cell deathmechanisms identified inFP-RMS following silencing of PAX3-FOXO1. We showed that cellsundergo intrinsic apoptosis in a NOXA-dependent manner uponreduction of PAX3-FOXO1protein levels. In accordance, we identifiedBH3-mimetics, specifically navitoclax, to efficiently enhance thismodeof cell death. Furthermore, we demonstrated a novel functionalinteraction between AURKA and the fusion protein and observedstrong synergy in vitro and in vivo with the AURKA inhibitor alisertiband navitoclax.

We established a functional link between PAX3-FOXO1 and theBH3-only protein NOXA.However, we were unable to identify exactlyhow the fusion protein regulates NOXA expression. Previously, it wasreported that PAX3-FOXO1 can upregulate basal Noxa levels in

Figure 4.

Aurora kinaseA regulatesMYCN in RMS cells andaffects MYCN protein stability. A, RMS cell lineswere transduced with lentivirus containing con-trol or Aurora kinase A (AURKA) shRNA (B andC) plasmids. Following selection, cellswere lysedand immunoblotted for MYCN. MYCN wasreduced in AURKA-silenced cells in comparisonwith control shRNA–treated cells. B, AURKAshRNA and control-treated cells were trans-fected with V5-tagged MYCN [either WT orT58A/S62A mutant (MT) plasmid], lysed after48 hours, and immunoblotted with V5 antibody.V5-MYCN (WT) was reduced in AURKA-silencedcells compared with controls, however, V5-MYCN (mutant) was not, indicating that AURKAcan regulate MYCN posttranslationally in RMScells by affecting protein stability. C, RMS-01 andRH4 were treated with increasing doses of ali-sertib and lysed after 48 hours to assess MYCNand PAX3-FOXO1 protein levels by immunoblot.

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http://r2.amc.nl


mouse myoblasts, which could prime tumor cells to apoptosis (40).Indeed, we also found that NOXA levels in tumor cells are higher thanin normal myoblasts. However, in chromatin immunoprecipitationsequencing data, we could not detect any binding of PAX3-FOXO1 tothe genomic PMAIP1 locus (41), excluding a direct role of the fusionprotein in NOXA regulation. This is supported by the observation thatNOXA levels even further increase when PAX3-FOXO1 levels dimin-ish. Because the cell lines used in our experiments are p53deficient (42),this also excludes a role of p53 in NOXA regulation as previouslydescribed (43). However, it is well known that NOXA expression canbe induced by a variety of cellular stresses such as hypoxia, DNAdamage, genotoxic, ER, or metabolic stress (44). If any of these, whichone might be responsible for the observed phenotype remains to beinvestigated.

Nevertheless, the observation that NOXA upregulation wasrequired for apoptosis of FP-RMS cells led to the identification ofnavitoclax as sensitizer. This is in line with the previous identificationof the BCL-XL inhibitor A-1331852 as sensitizer to chemotherapeu-tics (45). In addition, BCL-XL has been described as target gene of thefusion protein (46), which also explains the lower sensitivity of FP-RMS cells to venetoclax, targeting BCL-2. However, single-agentactivity is likely limited and therefore we searched for drugs capableto reduce fusion protein levels using conventional Western blotting.This led to the identification of multiple AURKA inhibitors thatalso reduced MYCN protein levels, a functionally very importantPAX3-FOXO1 target gene (35).

Previously, it was reported that fusion protein levels could bereduced by entinostat, an HDAC inhibitor (29). Conversely, in our

Figure 5.

Navitoclax and alisertib synergistically induce cell death in vitro. A, Synergy screen. Rh4 cells were incubated with each drug (Supplementary Table S1) at aconcentration of 500 nmol/L and additionally with either 800 nmol/L navitoclax or DMSO. After 48 hours, viability was assessed byWST-1 assay. Left y-axis, blackbars, compound; gray bars, compound plus navitoclax. Right y-axis, red bars, viability ratio (þnavitoclax/þDMSO). Top hits with a viability ratio < 0.7 are shown andare ranked according to viability of each drug alone. Red stars, Aurora kinase A inhibitors. Circle plot, 28 top hits were classified according to their target spectrum;red, Aurora kinase A inhibitors.B,Relative cell viability in percent after cross-titration of alisertib against navitoclax in Rh4 cells. Cell viability relative to DMSO controlwas measured after 48 hours. Color scheme: high viability, blue; low viability, red. Mean values of three independent experiments performed in duplicates aredepicted. C, BLISS synergy scores of Rh4 cells, indicated 6 PDX-derived primary cell cultures, human foreskin fibroblasts (HFF), and humanmyoblasts. Synergy wascalculated according to the Bliss independence model using the SynergyFinder WebApp (28). Positive values (red) indicate synergy, and negative values (green)indicate antagonism. D, Cell-cycle analysis of Rh4 cells treated with given concentrations of alisertib and additionally with either DMSO or 800 nmol/L navitoclax.After 24 hours, cells were stained with propidium iodide (PI), and cell cycle was analyzed by flow cytometry. Mean of two independent experiments.






screen for drugs affecting fusion protein levels, epigenetic regulators,like entinostat or SAHA, do not show a striking effect. However, thedrug concentrations used in our screen, as well as incubation times(0.1 mmol/L for 48 hours), are substantially lower than what was used

by Abraham and colleagues (2 mmol/L for 72 hours; ref. 29). It ispossible that these factors are the reasons for epigenetic regulators likeentinostat not showing strong effects in our screen (SupplementaryFig. S3B).

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Combination of navitoclax and alisertib reduces tumor growth in vivo. Rh4 or IC-PPDX-35 cells were injected s.c. into the flanks of NSGmice. After engraftment, micewere randomized and assigned to one of four treatment groups: vehicle, black; navitoclax only, gray (3d/week 80mg/kg); alisertib only, beige (5d/week 30mg/kg);navitoclaxþ alisertib combination, red.Micewere treated for 3weekswith the respective regimen through oral administration (see also Supplementary Fig. S7A) andsacrificedwhen tumors reached a size of 1,000mm3.A, Tumor growth of Rh4 cells in vivo. Black arrow, start of treatment; red box, treatment period. Per group: n¼ 6;error bars, SEM. B, Kaplan–Meier graph showing percent survival of different treatment groups. Mantel–Cox test for comparison of survival curves, �� , P¼ 0.001. C,Tumor growth of IC-PPDX-35 tumors. Black arrow, start of treatment; red box, treatment period. Per group: n¼ 5; error bars, SEM. D, Kaplan–Meier graph showingpercent survival.E,Histology of engrafted IC-PPDX-35 tumors after 1weekof treatmentwith either vehicle control (left plot) or combination of navitoclax (80mg/kg)andalisertib (30mg/kg; right plot).N¼ 3. Topplot, hematoxylin &eosin (H&E) staining. Bars, 20mm.Black arrows, apoptotic cells; red arrow,mitotic cell. Bottomplot,IHC staining against cleaved caspase-3. Bars, 50 mm. Representative images of sections from three different mice per group.

Ommer et al.





Strikingly, we identified a new phosphorylation site in PAX3-FOXO1 (serine 437) that is important for fusion protein stability.This site corresponds to S256 in WT FOXO1, where it regulatesnuclear exclusion and subsequent degradation (47), whereas phos-phorylation of S437 in PAX3-FOXO1 stabilizes the fusion protein.These seemingly opposing effects on protein stability might suggestthat protein turnover of WT versus the fusion protein is regulated bydifferent mechanisms, as recently also suggested for the EWS-FLI1fusion expressed in Ewing sarcoma (48). A similar observation hasalready been described for acetylation of K426 andK429 by the histoneacetyltransferase KAT2B that also stabilize PAX3-FOXO1 (49). Acet-ylation of the corresponding sites in WT FOXO1 results in phosphor-ylation at S256 and subsequent degradation (27). Hence, we mightspeculate that acetylated K426/K429 might contribute to stabilitythrough priming of the fusion protein for S437 phosphorylation.

In the past, clinical trials involving navitoclax or alisertib have beenchallenging halting the process of clinical development. Navitoclax isknown to increase the risk for thrombocytopenia due to its on-targeteffect (50). The advantage of our synergistic combination approach isthat the individual drug doses can be lowered, thereby potentiallyreducing unwanted effects of each individual drug. Indeed, in ourin vivo experiments, we did not observe complications when usingboth drugs in combination. Furthermore, recent interest in these drugsincreased, and both companies are currently recruiting to new clinicaltrials (www.clinicaltrials.gov).

Taken together, we identified a previously undescribed site inPAX3-FOXO1 that is important for fusion protein stability. WithAURKA inhibition, we found a therapeutic option to target this site inthe fusion protein while simultaneously also affecting MYCN levels(Supplementary Fig. S11A and S11B). It is likely that both fusionprotein and MYCN levels contribute to the inhibitory effects ofalisertib because positive feedback mechanisms have been describedregulating the expression of these two proteins (10, 12). Furthermore,by characterizing the exact mechanism of cell death associated with

loss of fusion gene expression, we were able to identify drugs thatenhance this mode of cell death. When used in such a rationalecombination, both drugs show a high degree of synergy bothin vitro and in vivo, in cell lines and an RMS PDX model. Thesefindings shed more light on a devastating disease and may offer noveltherapeutic options for the treatment of patients with alveolar RMS.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors’ ContributionsConception and design: J. Ommer, M. Wachtel, J. Shipley, B.W. Sch€aferDevelopment of methodology: J. Ommer, J.L. Selfe, M. Wachtel, D. SurdezAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. Ommer, J.L. Selfe, M. Wachtel, E.M. O'Brien, D. Laubscher,M. Roemmele, S. Kasper, O. Delattre, D. Surdez, G. Petts, J. ShipleyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Ommer, J.L. Selfe, M. Wachtel, D. Laubscher,O. Delattre, A. Kelsey, J. Shipley, B.W. Sch€aferWriting, review, and/or revision of the manuscript: J. Ommer, J.L. Selfe,M. Wachtel, A. Kelsey, J. Shipley, B.W. Sch€aferAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): D. Surdez, G. PettsStudy supervision: J. Shipley, B.W. Sch€afer

AcknowledgmentsThe authors wish to acknowledge the financial support from Swiss National

Science Foundation (310030_156923 and 31003A_175558), Childhood CancerResearch Foundation Switzerland, and Cancer League Switzerland (KLS-3868-02-2016).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 10, 2019; revised September 12, 2019; accepted December 18, 2019;published first December 30, 2019.

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2020;80:832-842. Published OnlineFirst December 30, 2019.Cancer Res Johannes Ommer, Joanna L. Selfe, Marco Wachtel, et al. Deathand Synergizes with Navitoclax to Induce Rhabdomyosarcoma Cell Aurora A Kinase Inhibition Destabilizes PAX3-FOXO1 and MYCN

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