Targeted Therapy based Combination Treatment in ... · Targeted Therapy–based Combination Treatment in Rhabdomyosarcoma Anke E.M.van Erp1,Yvonne M.H.Versleijen-Jonkers1,Winette

Review

Targeted Therapy–based Combination Treatmentin RhabdomyosarcomaAnke E.M. van Erp1, Yvonne M.H. Versleijen-Jonkers1,Winette T.A. van der Graaf1,2,and Emmy D.G. Fleuren3

Abstract

Targeted therapies have revolutionized cancer treatment;however, progress lags behind in alveolar (ARMS) and embry-onal rhabdomyosarcoma (ERMS), a soft-tissue sarcoma mainlyoccurring at pediatric and young adult age. Insulin-like growthfactor 1 receptor (IGF1R)-directed targeted therapy is one of thefew single-agent treatments with clinical activity in these dis-eases. However, clinical effects only occur in a small subset ofpatients and are often of short duration due to treatmentresistance. Rational selection of combination treatments ofeither multiple targeted therapies or targeted therapies withchemotherapy could hypothetically circumvent treatment resis-tance mechanisms and enhance clinical efficacy. Simultaneoustargeting of distinct mechanisms might be of particular interest

in this regard, as this affects multiple hallmarks of cancer atonce. To determine the most promising and clinically relevanttargeted therapy–based combination treatments for ARMS andERMS, we provide an extensive overview of preclinical and(early) clinical data concerning a variety of targeted therapy–based combination treatments. We concentrated on the mostcommon classes of targeted therapies investigated in rhabdo-myosarcoma to date, including those directed against receptortyrosine kinases and associated downstream signaling path-ways, the Hedgehog signaling pathway, apoptosis pathway,DNA damage response, cell-cycle regulators, oncogenic fusionproteins, and epigenetic modifiers. Mol Cancer Ther; 17(7); 1365–80.�2018 AACR.

IntroductionRhabdomyosarcoma is the most common type of soft-tissue

sarcoma (STS) observed in young patients with the mostfrequent subtypes being embryonal (ERMS) and alveolar rhab-domyosarcoma (ARMS). ERMS represents approximately 70%of childhood rhabdomyosarcoma and is most often observedin the head and neck region and genitourinary track. ARMS isseen in approximately 30% of rhabdomyosarcoma cases andusually occurs in the deep tissue of the extremities. The majorityof ARMS tumors are characterized by a fusion between PAX3 orPAX7 on chromosome 2 and FOXO1 on chromosome 13(�80%). The remaining 20% are fusion negative. Althoughgenerally ARMS have a poorer outcome compared with ERMS,fusion-negative ARMS show a genetic profile similar to ERMSand an equally favorable clinical outcome. Multimodality

treatment including surgery, chemotherapy, and radiotherapyhas increased the 5-year overall survival (OS) to approximately70%–90% for intermediate- and low-risk rhabdomyosarcoma,respectively. However, patients with high-risk rhabdomyosar-coma still have a 5-year OS of <40%. In addition, treatment-related toxicities severely decrease quality of life (1, 2). In anattempt to increase survival and improve quality of life, thefield of targeted therapy has gained substantial interest inrhabdomyosarcoma, and its potential is supported by variouslines of (pre)clinical research, which are mainly centered ontargeted therapies originally developed for other tumor types.In the clinic, however, intrinsic and acquired resistancemechanisms have emerged as common pitfalls in rhabdomyo-sarcoma treatment. As such, increasing evidence exists thatsingle-agent targeted therapy will not be sufficient to reachclinical efficacy in patients with rhabdomyosarcoma. The cur-rent hypothesis is that combination therapy could enhanceclinical efficacy and/or decrease treatment-associated toxicities.In this regard, simultaneous targeting of different mechanismsof action could be more effective as opposed to combininginhibitors of similar classes, as the characteristic hallmarks ofcancer illustrate that tumor progression is regulated by a widevariety of different processes.

To determine the most promising and clinically relevantcombination treatments for rhabdomyosarcoma, we reviewedthe preclinical and (early) clinical trial data addressing combi-nations of targeted therapies or targeted therapy combined withchemotherapy. We focused on the most common classes oftargeted therapies investigated in rhabdomyosarcoma to date,including those directed against receptor tyrosine kinases (RTK)and associated downstream signaling pathways, the Hedgehogsignaling pathway, apoptosis pathway, DNA damage response,cell-cycle regulators, fusion proteins, and epigenetic modifiers.

1Department of Medical Oncology, Radboud University Medical Center, Nijme-gen, the Netherlands. 2The Institute of Cancer Research, Division of ClinicalStudies, Clinical and Translational Sarcoma Research and The Royal MarsdenNHS Foundation Trust, Sutton, United Kingdom. 3The Institute of CancerResearch, Division of Clinical Studies, Clinical and Translational SarcomaResearch, Sutton, United Kingdom.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors:Winette T.A. van der Graaf, Division of Clinical Studies,The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust,15 Cotswold Road, SM2 5NG, Sutton, United Kingdom. Phone: 4402-0872-24448; Fax: þ31-24-36-15025; E-mail: [email protected]; andEmmy D.G. Fleuren, [email protected]

doi: 10.1158/1535-7163.MCT-17-1131

�2018 American Association for Cancer Research.

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RTKsRTKs are membrane-bound proteins involved in signal trans-

duction to the tumor cell. Activation by extracellular ligandbinding or genetic mutations can lead to constitutive activity.Intracellular signaling pathways, including the PI3K/AKT/mTOR,RAS/MEK/ERK, and JAK/STAT3 pathway, are subsequently acti-vated. Several RTKs have been identified as possible targets fortherapy in rhabdomyosarcoma, including the insulin-like growthfactor 1 receptor (IGF1R), anaplastic lymphoma kinase (ALK),platelet-derived growth factor receptors a and b (PDFGRa/b),VEGFR, EGFR, and the fibroblast growth factor receptor 4(FGFR4; Fig. 1A; ref. 3). Despite the promising preclinical effects,clinical efficacy is limited and observed in small subsets of pati-ents (4, 5). Combination treatment might enhance the clinicalefficacy of RTK-targeted therapies (Table 1).

IGF1R/ALK. One way of optimizing IGF1R treatment might beits combination with other (R)TK inhibitors. As coexpression ofthe RTKs IGF1R and ALK has been described in rhabdomyo-sarcoma, this may present a rational combination. In vitro,combined anti-IGF1R antibody R1507 and ALK inhibitorTAE684 treatment showed synergism in ARMS cell lines. InERMS, however, no enhanced effect was observed (6). In ARMS,the characteristic PAX3-FOXO1 protein can enhance IGF1Rand ALK transcription, possibly explaining this difference insensitivity. However, we, among others, could not find intrinsicALK activity in rhabdomyosarcoma cells (7–9). In addition, theantitumor effects observed with the ALK inhibitor ceritinibwere mostly explained by its capacity to inhibit IGF1R signaling(9). Combined treatment of ceritinib with the multikinaseinhibitor sorafenib showed synergistic effect in vitro (8). Inaddition, we observed high activity of the signaling protein Srcpost ceritinib treatment in both subtypes, and combined treat-ment of ceritinib and the Src inhibitor dasatinib was synergisticin vitro (9). Prior to our finding, increased activity of theSrc family tyrosine kinase (SFK) YES was observed in IGF1Rtreatment–resistant cell lines. Combination treatment of IGF1Rantibodies and SFK inhibitors led to superior in vitro apoptosisinduction and in vivo tumor growth reduction compared withthe monotherapies (10). One phase I/II trial is recruiting ARMSand ERMS patients to investigate the combined effects of theIGF1R antibody ganitumab and dasatinib (NCT03041701;Table 2). No results have yet been reported; however, basedon the consistent preclinical findings concerning IGF1Rtargeting and an increase in Src signaling, the results areeagerly awaited.

In addition to an increase in Src activity, ERMS showedenhanced PDGFRb activity as a resistance mechanism to IGF1Rtreatment. The combination of IGF1R- and PDGFRb-targetedtherapies increased growth inhibition in IGF1R-resistant ERMScell lines. Three PDGFRb inhibitors were tested, and combinedtherapy with pazopanib (VEGFR, PDGFR, c-KIT) or crenolanib(PDGFRa/b) was the most beneficial in vivo. The combinationsdelayed tumor growth compared with each monotherapy,although no complete tumor regressions were achieved (11).

Resensitization of IGF1R treatment–resistant tumor cellswas also examined by combinations of IGF1R ligand antago-nists and downstream signaling protein inhibitors. Insulin-likegrowth factor binding proteins (IGFBP) regulate binding ofinsulin-like growth factor 1 and -2 (IGF1/2) to IGF1R. In ARMS,IGF1R-resistant cell lines showed reduced IGFBP2 expression

and a limited decrease of downstream AKT activity uponIGF1R antibody treatment. IGF1R antibodies combined withrecombinant IGFBP2, the PI3K inhibitor BKM130, or themTOR inhibitor Ku-0063784 resensitized the resistant celllines to IGF1R targeting (12). The possible low bioavailabilityof recombinant IGFBP2 could affect the clinical potential ofthis particular combination treatment. Clinical trials didshow a partial response (PR) in a patient with IGF1R-positivesoft-tissue sarcoma and stable disease (SD) in 2 of 11 pediatric,adolescent, and young adult (AYA) patients with rhabdomyo-sarcoma for the combination of IGF1R inhibitors and mTORinhibitors (Table 2; refs. 13–16). This shows that only a smallgroup responded to treatment and one study showed that theaddition of temsirolimus led to increased toxicity without aclear increase in efficacy in most patients (15). Nevertheless,these data do show that combined targeted therapy can over-come primary treatment resistance and suggest that, whengiven simultaneously, might delay or prevent treatment resis-tance altogether.

Because chemotherapy remains fundamental in rhabdomyo-sarcoma treatment, combinations with chemotherapy havebeen investigated. In young patients with ARMS and ERMS,the combination of the IGF1R antibody cixutumumab withconventional chemotherapy was compared with the combina-tion of temozolomide with conventional chemotherapy. Thecombination with cixutumumab led to a higher percentageof patients reaching an 18-month event-free survival (EFS;cixutumumab 68% vs. temozolomide 39%; NCT01055314;Table 2). Of note, the combination with cixutumumab hadmore reports of high-grade toxicity compared with the combi-nation with temozolomide. High-grade toxicities were, how-ever, only observed in a very small group (3/97, 3%), leavingthe combination with cixutumumab preferential to the combi-nation with temozolomide (17). Although preclinical researchand limited clinical research suggests that combinations oftargeted and cytostatic agents could render success, phase Istudies should first investigate the optimal dose and scheduleof new combinations in clinical practice.

VEGFR. VEGFs bind to VEGFRs and induce angiogenesis. Thismakes both VEGFs and VEGFRs a possible target for treatment.The camptothecin analogue namitecan was shown to reduceangiogenesis in an ERMS mouse model. Moreover, combinedtreatment of low-dose namitecan with either the VEGF anti-body bevacizumab or the VEGFR inhibitor sunitinib led to anenhanced tumor growth reduction compared with the mono-therapies. Sunitinib was in favor of bevacizumab, possibly dueto the effect of camptothecins on VEGF expression or themultikinase inhibition of sunitinib (18). VEGF can also betargeted via inhibition of heparanase. Heparanase is an enzymenecessary to generate heparin sulfate–bound growth factors,including VEGF. Heparanase activity is increased in rhabdo-myosarcoma and combination treatment of the heparanaseinhibitor SST0001 and bevacizumab or sunitinib reducedangiogenic growth factor expression and decreased cellularinvasion in vitro. In vivo SST0001 monotherapy effectivelydecreased ARMS and ERMS tumor volumes. No further exam-ination of the combination was performed in vivo, precludingexamination of the clinical potential of combinations withSST0001 (19). Clinical trials have not (yet) shown convincingeffects of anti-VEGF(R) combination treatments in young

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Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1366



Figure 1.

Overview of the cellular processes used as targets in the targeted therapy–based combination treatments in ARMS and ERMS. A, Membrane-boundgrowth factor receptors IGF1R, ALK, PDGFR, FGFR4, EGFR, VEGFR, Patched 1 (PTCH1), SMO, and TRAILR1/2; ligands IGF1/2, VEGF and (Sonic, Indian,Desert) Hh; intracellular signaling proteins of the PI3K/AKT/mTOR, JAK/STAT3, RAS/MEK/ERK, Hh and apoptosis pathway (4E-BP1, eukaryotic translationinitiation factor 4E-binding protein 1; eIF4E, eukaryotic translation initiation factor 4E; JAK, Janus kinase; FADD, Fas-associated protein with deathdomain; CASP, caspase; RIP1, receptor-interacting serine/threonine-protein kinase 1; BID, BH3 interacting-domain death agonist; BAX, Bcl-2-associated Xprotein; BAK, Bcl-2 homologous antagonist killer; BCL-2, B-cell lymphoma 2; MCL-1, induced myeloid leukemia cell differentiation protein; BCL-XL, B-celllymphoma-extra large; Smac, second mitochondria-derived activator of caspases; Cyt C, cytochrome C, IAP, inhibitor of apoptosis protein). B andC, Intranuclear processes: DNA damage response (DDR) (PARP), the epigenome and the cell cycle that are used as therapeutic targets in (pre)clinicalcombination treatments in ARMS and ERMS.

Targeted Therapy–based Combination Treatment in RMS

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Table 1. Preclinical targeted therapy–based combination treatments in ARMS and ERMS

Category Target(s) CombinationaSubtypesincluded In vivo

Effect combination/effectmonotherapy Ref.

RTKsIGF1R IGFR þ ALK R1507 þ TAE684 ARMS

ERMSNo CI � 1.0 in ARMS (Rh41, Rh30) cell

lines(6)

ALK (IGF1R) þ multi-kinase Ceritinib þ sorafenib ARMSERMS

No CI < 1.0 in ERMS (RD) and ARMS(Rh30) cell lines (ceritinib þ�10 mmol/L sorafenib)

(8)

ALK (IGF1R) þ Src Ceritinib þ dasatinib ARMSERMS

No CI < 1.0 in ARMS (Rh41, Rh30) andERMS (RD, Rh18) cell lines

(9)

IGF1R or IR/IGF1R þ YES MK0646 þ AZD0530 ARMS Yes In vitro: Increased apoptosis (10)BMS754807 þ AZD0530 ERMS In vivo: Increased tumor reductionR1507 þ AZD0530R1507 þ dasatinibb

IGF1R þ PDGFRb R1507 þ pazopanib ARMS Yesc In vitro: Re-sensitization to anti-IGF1R treatment

(11)R1507 þ crenolanibb ERMS

In vivo: Slight increased delay tumorgrowth, no complete regression

R1507 þ imatinib

IGF1R þ PI3K or mTORC1/2 R1507 þ recombinant IGFBP2 ARMS No Re-sensitization to anti-IGF1Rtreatment

(12)R1507 þ buparlisibR1507 þ Ku-0063794

VEGFR VEGF(R) þ chemotherapy Bevacizumab þ namitecan ERMS Yes In vivo: Low dose namitecanenhanced tumor growthreduction

(18)Sunitinib þ namitecan

Heparanase þ VEGF(R) SST0001 þ bevacizumab ARMS No Reduced angiogenic factorexpression

(19)

SST0001 þ sunitinib ERMS Reduced cellular invasionEGFR EGFR þ chemotherapy Cetuximab þ dactinomycin ARMS

ERMSNo CI < 1.0 in ARMS (Rh30) and ERMS

(RD) cell lines with increasedapoptosis

(22)

EGFR (drug conjugate) EGFR-conjugated immunotoxin(exotoxin A)

ERMS No N.A. (23)

EGFR (drug conjugate) EGFR-conjugated immunotoxin(human granzyme B)(þchloroquine)

ERMS No N.A. (chloroquine increasedpotency)

(24)

FGFR FGFR4 þ IGF1R BGJ398 þ AEW541 ARMS No CI < 1.0 in ARMS (RMS13) cell line (29)PDGFRa Multi-kinase þ Src Imatinib þ PP2 (murine) No Imatinib þ PP2: Enhanced cell

viability reduction(31)

Sorafenib þ PP2 ARMSSorafenib monotherapy: Most

effective in reducing cell viability.Effective in absence of PDGFRa.

Sorafenib þ PP2: No added effectDownstream signaling pathwaysPI3K/AKT/mTOR PI3K þ IGF1R or mTOR or

MEKBuparlisib þ AEW541 ARMS No CI < 1.0 in ARMS (SJCRH30) and

ERMS (RD) cell lines(36)

Buparlisib þ rapamycinc

Buparlisib þ trametinibc ERMSPI3K þ MEK PI103 þ U0126 ARMS

ERMSNo CI < 1.0 in ARMS (Rh30, RMS13) and

ERMS (RD) cell lines(37)

PI3K þ MEK AZD8055 þ selumetinibb ARMS Yes In vitro:CI < 1.0 in ARMS (Rh30) andERMS (RD, RMS-YM) cell lines

(38)ZSTK474 þ selumetinibc ERMS

In vivo:AZD8055: Enhanced

reduction downstream signalingBEZ235 þ selumetinibb,c

Increased toxicityBEZ235: No added effect

RAS/MEK/ERK MEK þ chymotrypsin-likeserine protease

PD98059 þ TPCK ERMS Yesd In vitro: Increased reduction cellproliferation

(44)

In vivo: Enhanced reduction tumorgrowth

MEK U0126 þ radiotherapy ARMS No In vitro: ERMS: Enhanced reductionsphere culture

(45)ERMS

ARMS: No added effectJAK/STAT3 STAT3/GP160 þ MEK LY5 þ doxorubicin ARMS No Increased inhibition STAT3 activity (46)

LY5 þ cisplatin Increased reduction of cellmigrationLY5 þ selumetinib

Increased apoptosisBazedoxifine þ doxorubicinBazedoxifine þ cisplatinBazedoxifine þ selumetinib

(Continued on the following page)

van Erp et al.




Table 1. Preclinical targeted therapy–based combination treatments in ARMS and ERMS (Cont'd )



Hedgehog signalingHedgehog GLI1–2 þ mTOR GANT61 þ temsirolimus ARMS No Induction cell cycle arrest (49)

GANT61 þ rapamycin ERMS Increased apoptosisGANT61 þ vincristine

GLI1-2 þ PI3K/mTOR GANT61 þ PI103b ARMS Yese In vitro: Enhanced apoptosis withreduced downstream signaling

(50)GANT61 þ BEZ235 ERMS

In vivo: Decreased clonogenicsurvival, sphere formation andtumor growth

GANT61 þ GDC0941GANT61 þ RAD001GANT61 þ AZD8055

GLI1 þ chemotherapy ATO þ vincristine ARMS No CI < 1.0 in ARMS (Rh30) and ERMS(RD) cell lines

(51)ATO þ vinblastine ERMSATO þ eribulin

GLI1 þ SMO ATO þ itraconazole ARMSERMS

No Limited added effects in spheroidculture

(52)

GLI1 þ GSK3 ATO þ lithium chloride ARMSERMS

No Limited added effects in spheroidculture

(53)

Apoptosis pathwayApoptosis TRAILR1 of TRAILR2 þ IAP mapatumumab þ IAPi

#2f(TRAILR1)ARMS No TRAILR1: No added effect (54)

mapatumumab þ IAPi #3fERMS TRAILR2: Increased reduction cell

viabilitylexatumumab þ IAPi #2(TRAILR2)

lexatumumab þ IAPi #3Survivin þ chemotherapy YM155 þ cisplatin ERMS Yes In vitro/in vivo: Increased cell

viability and caspase-3 activity(55)

Despite increase caspase 3 activity,no increase in apoptotic cells

mTOR þ Bcl-2/BCL-xL/BCL-w or chemotherapy

AZD8055 þ ABT737 ARMS No ABT737: CI <1.0 in ARMS (RMS13,Rh30) and ERMS (RD, TE671) celllines

(56)AZD8055 þ doxorubicin ERMS

Chemotherapy: No added effectsAZD8055 þ vincristineAZD8055 þ dactinomycin

DNA damage response (DDR)PARP1 PARP1 and/or tdp1 siRNA

with or withoutchemotherapy

Rucaparib þ irinotecan ARMS No PARP/tdp1 siRNA þ irinotecan:Increased irinotecan sensitivity

(59)Olaparib þ irinotecan ERMS

Combination tdp1 siRNA: Addedeffect on cell viability

Veliparib þ irinotecantdp1 siRNA þ irinotecanRucaparib þ tdp1 siRNA

PARP1 þ chemotherapy Olaparib þ carboplatin ARMS No Irinotecan, melphalan,doxorubicin: CI < 1.0 in ARMS(SJCRH30) and ERMS (RD) celllines

(60)Olaparib þ SN38 ERMS

Carboplatin, vincristine: CI >0.5and �1.0 in ARMS (SJCRH30)and ERMS (RD) cell lines

Olaparib þ vincristine

Olaparib þ melphalanOlaparib þ doxorubicin

PARP1 þ chemotherapy Talazoparib þ temozolomideh ARMS Yesk In vitro:Temozolomide:potentiating anti-tumor effects inARMS (Rh41, Rh30) and ERMS (RD,Rh18) cell lines

(61)

Talazoparib þ topotecan ERMS

Topotecan: No added effectsIn vivo: 2/3 models showedtreatment response1/3 models showed maintained CR

Cell cyclePLK PLK1 catalytic domain þ

chemotherapyBI2536 þ vincristineh ARMS Yes In vitro:

Vincristine, vinblastine,vinorelbine: CI < 1.0 in ARMS

(Rh30) and ERMS (RD) cell lines

(65)

BI2536 þ vinblastine ERMS

Doxorubicin, paclitaxel: CI > 1.0 inARMS (Rh30) andERMS (RD) celllines

BI2536 þ vinorelbine

In vivo: Reduced tumor growthwhich remained stable for 56days

BI2536 þ doxorubicinBI2536 þ paclitaxelVolasertib þ vincristineh

Volasertib þ vinblastineVolasertib þ vinoelbineVolasertib þ doxorubicinVolasertib þ paclitaxel

PLK1 catalytic domain þchemotherapy

Volasertib þ vincristine ERMS No Vincristine: CI � 1.0 in ERMS(RMS-1) cell line

(66)Volasertib þ etoposide

Etoposide: CI > 1.0 in ERMS (RMS-1)cell line

(Continued on the following page)





Table 1. Preclinical targeted therapy–based combination treatments in ARMS and ERMS (Cont'd )



PLK1 catalytic domain orpolo-box domain þchemotherapy

BI2536 þ eribulinh ARMS Yesj Catalytic domain:In vitro: CI < 1.0 inERMS (RD, TE381.T) CI 0.5-1 and� 1in ARMS (RMS13, Rh30) cell lines.

(67)

Poloxin þ vincristine

l

ERMS

In vivo: Reduction tumor growthPolo-box domainIn vitro: CI 0.5-1.0 and� 1.0 in ERMS(RD) cell line

Wee1 Wee1 þ proteasome ormulti-kinase orchemotherapy

AZD1775 þ bortezomib ARMS No Cabozantinib and bortezomib:Most effective in combinationwith AZD1775

(70)AZD1775 þ cabozantinib ERMSAZD1775 þ cyclophosphamideAZD1775 þ irinotecanAZD1775 þ etoposideAZD1775 þ dactinomycinAZD1775 þ virorelbine

Wee1 þ chemotherapy AZD1775 þ irinotecan þvincristine

(O-PDX)ARMSERMS

Yesk Increased tumor responsecompared to monotherapyAZD1775 or combinedchemotherapy

(71)

CDK CDK þ IGF1R Palbociclib þ BMS754807 RMSp No CI not available, synergistic effectsmentioned

(73)

CDK þ Wee1 orchemotherapy

Palbociclib þ AZD1775 ERMS No AZD1775: CI < 1.0 in ERMS (RD) cellline

(74)Palbociclib þ doxorubicin

Doxorubicin: CI > 1.0 in ERMS (RD)cell line

Fusion protein PAX3-FOXO1 RGB-LRP-P3Fo ARMS Yes N.A. (75)EpigenomeHistone modification HDAC þ chemotherapy Vorinostat þ doxorubicin ARMS No Increased reduction cell viability

and increased apoptosis(77)

Vorinostat þ etoposide ERMSVorinostat þ cyclophosphamideVorinostat þ vincristine

HDAC þ chemotherapy Quisinostat þ doxorubicing ARMS Yesh,j In vitro: Increased reduction cellviability and increased apoptosis

(78)

Quisinostat þ etoposide ERMSIn vivo: Enhanced reduction tumor

growthQuisinostat þ vincristineQuisinostat þ cyclophosphamideQuisinostat þ dactinomycin

HDAC þ chemotherapy Vorinostat þ cisplatin ERMS No Increased reduction cell viabilityand increased apoptosis

(79)Valproic acid þ cisplatin

HDAC þ multi-kinase orHSP90 or nucleosidereverse transcriptase

Vorinostat þ sorafenib ERMS No Sorafenib: CI < 1.0 in ERMS (RD18)cell line

(80)Vorinostat þ 17-DMAG

17-DMAG/abacavir: CI > 1.0 inERMS (RD18) cell line

Vorinostat þ abacavir(Sorafenib þ 17-DMAG)

(Sorafenibþ 17-DMAG: CI < 1.0 inERMS (RD18) cell line)

HDAC þ SMO or mTORC1 þCOX2 or chemotherapy

Valproic acid þ vismodegib(SMO) þ Atorvastatin

ARMSPDX

Yesl Etinostat þ docetaxel vs.etinostat: 65% tumor regressionvs. 43% tumor regression

(81)

Valproic acid þ metformin(mTOR) þ celecoxib (COX2) All other combination: PD

Entinostat þ docetaxelLSD þ HDAC GSK690 þ Quisinostat ARMS No CI < 1.0 in ARMS (Rh30) and ERMS

(RD) cell lines(82)

GSK690 þ Vorinostat ERMSEx917 þ Quisinostat

DNA methylation DNA demethylation þ CTLactivity

50 aza-20-deoxtidine (DAC)þ CTL ARMSERMS

No DAC increased sensitivity to CTLcytotoxicity

(85)

Abbreviations: CI, combination index; CI< 1.0, synergism;CI¼ 1.0, additive effect; CI> 1.0, antagonism; IR, insulin receptor; IAPi, inhibitor of apoptosis protein inhibitor;N.A., not applicable; SN38, irinotecan active metabolite.aPubChem ID available in Supplementary Table S1.bCombination tested in vivo.cOnly tested ERMS.dZebrafish model.eChicken embryo model.fCompound not further specified.gPubChem ID available in Supplementary Table S1.hCombination tested in vivo.iOnly tested ERMS.jChicken embryo model.kOnly ARMS.lOnly in vivo examination.mLiposome-protamine-siRNA particles specifically directed against PAX3-FOXO1 fusion.nSubtype not specified.

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patients with rhabdomyosarcoma. Bevacizumab combinedwith standard chemotherapy did not significantly increasethe median EFS in patients with rhabdomyosarcoma(NCT00643565; Table 2; ref. 20). The combination of bevaci-zumab with sorafenib and low-dose cyclophosphamide didlead to a PR in a patient with rhabdomyosarcoma. However,only 2 patients with rhabdomyosarcoma were included in thestudy, making the clinical efficacy of this combination inpatients with rhabdomyosarcoma difficult to determine (21).

EGFR. Similar to IGF1R and VEGF(R), combined therapy of theanti-EGFR antibody cetuximab and the chemotherapeutic dacti-nomycin was superior to dactinomycin treatment alone in EGFR-expressing ARMS and ERMS cell lines (22). Despite the fact thatEGFR overexpression is present in 37%–76% of ERMS and 16%–

50%of ARMS (23), no in vivo or clinical trials have been describedfor EGFR-based combination treatment in rhabdomyosarcoma;hence, no solid conclusions can be drawn regarding the potentialof this combination in the clinic. A novel way of targeting EGFR incombination with cytotoxic compounds was recently introduced,by creating antibody–drug conjugations. Niesen and colleaguesused single-chain fragment variables (scFv) with high tumorpenetration capacities in combination with truncated exotoxinA to create immunotoxins (IT) based on the anti-EGFR antibodiescetuximab and panitumumab. EGFR binding led to the internal-ization of the compound and reduced cell viability in a number ofEGFR-expressing cell lines. Further analysis showedbinding of theIT to EGFR-expressing rhabdomyosarcoma cell lines and reducedcell viability with induction of apoptosis. Moreover, the IT wasshown to bind to EGFR-expressing rhabdomyosarcoma tumortissue (23). Niesen and colleagues more recently developed fullyhuman cytolytic fusion proteins (hCFP) by conjugating serineprotease granzyme B to EGFR scFvs. hCFPs were shown to besuperior to ITs since the generation of neutralizing antibodiesagainst the bacterial toxins was prevented. EGFR-directed hCFPsshowed similar effects as the IT, including binding to rhabdo-myosarcoma tumor tissue, and the effects were increased whencombinedwith potency-enhancing chloroquine (24). Despite thelack of in vivo experiments and clinical trials in patients withrhabdomyosarcoma, these hCFPs remain an interesting com-pound, especially because in vivo experiments with ITs in breastcancer xenografts, and a phase I study with anti-CD22 immuno-toxinmoxetumomab pasudotox in childhood acute lymphoblas-tic leukemia showed good tumor penetration and well-toleratedtoxicity levels, respectively (25, 26).

FGFR4. Similar to IGF1R, FGFR4 is a transcriptional target ofthe PAX3-FOXO1 fusion protein leading to increased proteinexpression (27). In rhabdomyosarcoma, FGFR4 targetingis expected to be most effective in those tumors expressingactivated FGFR4 due to amplification or mutation (28).Amplification is more likely to occur in ARMS as a result ofthe PAX3-FOXO1 fusion protein regulating FGFR4 expression.Indeed, higher activated FGFR4 expression levels were observedin ARMS as compared with ERMS cell lines (7). In contrast,whole-exome/transcriptome sequencing revealed activatedFGFR4 mutations in 4 of 60 patients with rhabdomyosarcoma,all of which were ERMS (28). The only study describing acombination treatment with anti-FGFR4 therapy showed thatFGFR4 activity functions as a compensatory mechanism to theeffects of combined IGF1R and PI3K/mTOR inhibitor treatment

in ARMS and ERMS cell lines. In addition, combined IGF1R andFGFR4 inhibitor treatment showed synergism in an ARMS cellline (29). This suggests that combinations of IGF1R and/orPI3K/mTOR and FGFR4 inhibitors might be able to preventacquired treatment resistance. The Pediatric MATCH study is anongoing trial, in which the pan-FGFR inhibitor JNJ-42756493is tested in pediatric patients with relapsed and refractoryadvanced solid tumors, including STS (NCT032107140). Nocombination studies with an FGFR4 inhibitor are currently inthe clinic for rhabdomyosarcoma.

PDGFRa. Expression of both PDGFRa and PDGF ligands hasbeen described in rhabdomyosarcoma tumors (30). However,single-agent treatment ultimately leads to treatment resistance.Imatinib-resistant murine ARMS cell lines no longer showed adecrease of PDGFRa activity, and demonstrated increased Srcactivity following imatinib (Abl, c-KIT, PDGFR) treatment. Com-bination therapy of imatinibwith the SFK inhibitor PP2 enhancedcell viability reduction in the resistant cell line. However, thiscombined treatment was not more effective than monotherapywith the PDGFR/RAF inhibitor sorafenib. Both in absence andpresence of PDGFR, sorafenib reduced cell viability more effec-tively than imatinib and/or PP2. A similar reduction in cellviability was seen in both na€�ve and imatinib-resistant cell linesfollowing sorafenib treatment. This suggests that PDGFRa activityis not as important for cell viability as RAF activity, questioningthe importance of PDGFRa targeting in ARMS (31). One clinicaltrial compared the combination of the multikinase inhibitorpazopanib (VEGFR, PDGFR) with the MEK inhibitor trametinibto pazopanib monotherapy in advanced STS. One sinonasalERMS was included, which showed a PR to the combined treat-ment. However, this patient was diagnosed with a PIK3CA E542Kaberration and previously progressed on PI3K inhibitor mono-therapy. Resistance to PI3K treatment can be related to an increasein MEK activity, the specific target of trametinib; thus, the PRmight not necessarily be linked to the combination with pazo-panib and might merely be the result of the administration oftrametinib (32). This again questions the influence of PDGFRainhibition on the observed effect. Nevertheless, the anti-PDGFRaantibody olaratumab has gained FDA approval for treatment ofSTS and olaratumab combined with doxorubicin showed anenhanced OS compared with doxorubicin alone in a group ofmixed STS. No inclusion of rhabdomyosarcoma was mentionedand PDGFRa expression did not correlate with outcome (33–35).The ANNOUNCE trials will further examine the use of olaratu-mab combined with chemotherapeutics in a larger group of STS(NCT02451943; NCT02659020). Results are not yet available,leaving the clinical efficacy of olaratumab in patients with rhab-domyosarcoma unknown.

Downstream signaling pathwaysPI3K/AKT/mTOR. The PI3K/AKT/mTOR pathway shows aber-rant activation in rhabdomyosarcoma, either by mutations inthe PIK3CA gene or by high levels of growth factor signaling.PI3K/AKT/mTOR signaling promotes gene transcription, cellgrowth, metabolism, cell motility, and invasion (Fig. 1A).Combination treatment of the PI3K inhibitor buparlisib withthe IGF1R inhibitor AEW541, the mTOR inhibitor rapamycin,or the MEK inhibitor trametinib showed synergism in vitro (36).Other MEK inhibitors (including selumetinib) showed similarsynergism with multiple PI3K/mTOR inhibitors (37, 38).





In vivo, the most effective treatment was the mTOR inhibitorAZD8055 combined with selumetinib, showing an enhancedreduction in downstream protein activity. This combinationwas hence suggested for further clinical evaluation (38).Clinical trials did find an increased efficacy of dual PI3K andMEK inhibition compared with the monotherapies in patientswith advanced cancer; however, this was at the cost of increasedtoxicity (39). Combination treatment based on inhibitingthese two main downstream signaling pathways might there-fore not be feasible in patients with rhabdomyosarcoma. Thecombination of mTOR inhibitors with chemotherapy mighthave more potential, as combinations with liposomal doxoru-bicin, irinotecan, temozolomide, vinblastine, or cyclophospha-mide and topotecan were well tolerated in pediatric, AYApatients (40–43). Two clinical studies reported a response ina small subset of patients with rhabdomyosarcoma (41, 42).

RAS/MEK/ERK pathway. Similar to PIK3CA, RAS mutations arepresent in a subset of ERMS leading to higher activity of thepathway (28). Both tyrosine and serine residues are phosphory-lated andmonotherapy with theMEK inhibitor PD98059 and thechymotrypsin-like serine protease inhibitor TPCK significantlydelayed tumor growth in a KRAS-mutated rhabdomyosarcomazebrafish model without affecting normal behavior and growth(44). The monotherapies reduced proliferation in an NRAS-mutated ERMS cell line, but did not induce apoptosis. Combi-nation of suboptimal concentrations significantly reduced in vitrocell proliferation and in vivo tumor growth, indicating that thiscombination could be of interest for RAS-mutated rhabdomyo-sarcoma. However, with the above-mentioned downstream pro-tein inhibitor combination in mind, the pharmacokinetics andtreatment-related toxicities should be closely monitored.

The inhibition of the RAS/MEK/ERK pathwaywas also tested incombination with radiotherapy. Cancer stem cells (CSC) areinvolved in self-renewal and migration of the tumor, and highexpression of CD133 is a CSC marker in ERMS. CD133-positiveERMS spheres treated with the MEK inhibitor U0126 showedreduced sphere formation and combined treatment with radio-therapy enhanced antitumor effects. This suggests that MEK isinvolved in ERMS CSCs and that CSC may be vulnerable tocombined radiotherapy and MEK treatment (45). These are,however, preliminary data; thus, further research is necessarybefore any potential clinical implications can be made.

JAK/STAT3 pathway. Persistent STAT3 activity has been reportedin rhabdomyosarcoma andmonotherapy with the STAT3-specificLY5 or the upstream STAT3 GP130 inhibitor bazedoxifene led todecreased cell migration and induced apoptosis in pSTAT3-pos-itive ARMS in vitro. The effects of both compounds could beenhanced by the addition of doxorubicin, cisplatin, or the MEKinhibitor AZD6244 (46). These results emphasize again thatcombinations of downstream protein inhibitors with either che-motherapy or other downstream inhibitors can be very capable ofimproving antitumor effects as compared with monotherapies.These are encouraging findings, although STAT3 inhibition com-bined with doxorubicin or cisplatin requires further in vivo eval-uation to determine whether clinical evaluation is worthwhile.

Hedgehog signalingSeveral well-known developmental pathways are involved in

tumorigenesis, including the Hedgehog (Hh) signaling pathway.

Hh signaling is involved in embryogenesis and ligand bindingleads to GLI transcription factor (GLI) activation and subse-quent target gene transcription. GLIs can also be activated by thePI3K/AKT/mTOR pathway, and a crosstalk between both path-ways has been identified in several tumor types. The subsequentGLI-1 and -2 activity has been shown to have oncogenic con-sequences (Fig. 1A; ref. 47).

Hh signaling can be constitutively active in rhabdomyosar-coma and the GLI1/2 inhibitor GANT-61 significantly reducedcell growth in rhabdomyosarcoma xenograft models, althoughno complete responses (CR) were achieved. Combined treat-ment of GANT-61 with temsirolimus, rapamycin, or vincristineincreased these effects, with a preference for temsirolimus overrapamycin (48, 49). Similar effects were observed in primaryrhabdomyosarcoma cells, in which multiple PI3K and/ormTOR inhibitors combined with GANT-61 led to enhancedapoptosis. In addition, combination treatment of GANT-61with the PI3K inhibitor PI103 showed a decreased clonogenicsurvival and growth in vivo. The combination was tested in achicken embryo model, making additional in vivo examinationin a fully formed organism necessary before a translation to theclinic can be made (50). Arsenic trioxide (ATO), an activecomponent of Chinese medicine with anti-GLI1/2 effects, incombination with vincristine, vinblastine, and eribulin alsoshowed synergism in both rhabdomyosarcoma subtypes in vitro(51). In addition, the dual inhibition of GLI1/2 and Hh-relatedprotein smoothened (SMO) or glycogen synthase kinase 3(GSK3) led to a significant reduction in colony formation inboth ARMS and ERMS cells. However, 3D-spheroid culture, as abetter representation of the in vivo situation, showed limitedadditive effects of these combinations (52, 53). The currentin vitro and in vivo models do not allow for direct translation ofthese combinations to a clinical setting. However, with addi-tional in vivo studies in more complex (mouse) models, thecombination of Hh inhibitors with either chemotherapy orPI3K/AKT/mTOR inhibitors could have potential for rhabdo-myosarcoma treatment.

Apoptosis pathwayMost targeted therapies are capable of inducing apoptosis.

However, some treatments specifically activate the apoptosispathway. Apoptosis is triggered via an extrinsic death receptorpathway and the intrinsic mitochondrial pathway. TNF-relatedapoptosis-inducing ligands (TRAIL) activate membrane-boundTRAIL receptors (TRAILR) and subsequently activate a caspasecascade or induce intrinsic mitochondrial pathway activation.Upon activation of the intrinsic pathway, mitochondrial cyto-chrome c, and Smac are released into the cytosol. Cytochrome csubsequently induces the activation of caspase-9, whereas Smacantagonizes the inhibitor of apoptosis (IAP) proteins (includingsurvivin; Fig. 1A; ref. 54).

TRAILR1- and TRAILR2-specific agonistic antibodies, eitheralone or in combination with IAP inhibitors, were examined inboth rhabdomyosarcoma subtypes. TRAILR1 and IAP inhibitormonotherapies were not effective. TRAILR2 therapy did show adose-dependent cell viability reduction, although this was inde-pendent of the level of TRAILR2 present in the cell line. TheTRAILR2 effects could be enhanced by addition of IAP inhibitorsin both ARMS and ERMS (54).

A specific survivin inhibitor, YM155, was recently testedas a monotherapy and in combination with cisplatin in

van Erp et al.




ERMS. YM155 reduced survivin levels and cell viability in vitro.Both in vitro and in vivo, combination treatment with cisplatinled to an enhanced effect on cell viability and caspase levels, butapoptosis only slightly increased as compared with cisplatinmonotherapy. No examination of YM155 in the ARMS subtypewas performed, leaving its use in ARMS to be evaluated (55).

Members of the antiapoptotic Bcl-2 family are overexpressed inrhabdomyosarcoma (Fig. 1A). Combined targeting of the Bcl-2family inhibitor ABT737 and the mTOR inhibitor AZD8055showed synergy in rhabdomyosarcoma cell lines. The addedeffects of AZD8055 with ABT737 were dependent on the inhibi-tion of bothmTOR complexes and a decrease inMCL-1 levels. Ofnote, combination of AZD8055 with chemotherapy was notsynergistic (56).

All of the abovementioned studies suggest targeting of apo-ptosis pathway–associated proteins to be a potential therapeuticoption. However, only one study examined in vivo effects andfurther research is necessary. Nonetheless, the combination ofanti-BcL-2 and mTOR inhibitors does give a good example of thepotential of a combination treatment that targets multiple pro-cesses in the tumor cell at once. Through the inhibition ofmultiple, distinct cellular processes, we might be able to generatea more robust antitumor effect without having to resort tocombinations with systemic chemotherapy. One clinical trial iscomparing the combination of a TRAILR2 agonist and chemo-therapy with TRAILR2 and an anti-IGF1R antibody in adult solidtumors, including sarcomas (NCT01327612; Table 2). This trialmight give more insights into the clinical efficacy of targetingmultiple cellular processes simultaneously and whether it ispreferential to combinations with systemic chemotherapy.

The abovementioned studies all describe targets consisting ofmembrane-bound growth factor receptors and associated intra-cellular signaling proteins. However, other intracellular processessuch as DNA damage repair, cell-cycle regulation, and geneexpression regulation are likewise targets for treatment (Table 1;Fig. 1B and C).

DNA damage response (DDR): PARP1The DNA damage response (DDR) plays a crucial role in

the defense against the deleterious effects of DNA damage(57, 58). Key to the DDR is PARP1. PARP1 is involved insingle-strand break repair, where its binding to damaged DNAleads to poly (ADP-ribose) (pADPr) chain synthesis and recruit-ment of repair proteins. pADPr chains are also involved in PARP1release from the DNA to ensure access of repair proteins to thedamaged site (Fig. 1B). In rhabdomyosarcoma, monotherapy ofthe PARP inhibitor olaparib showed intermediate effects on cellviability. Combination of PARP inhibitors with multiple che-motherapeutics showed enhanced in vitro effects for combinedtreatment with irinotecan, melphalan, doxorubicin, and temo-zolomide (59–61). Lower synergism was seen for the combina-tionwith carboplatin or vincristine, and addition of topotecandidnot enhance effects (60, 61). Moreover, the PARP inhibitortalazoparib combined with temozolomide showed response in2 of 3 ARMSmodels in vivo, and onemodel showed amaintainedCR until the end of the study (61). In addition, PARP and DNArepair enzyme tyrosyl-DNA phosphodiesterase (tdp1) can form aDNA repair complex. Genetic knockdown of tdp1 combinedwithirinotecan or the PARP1 inhibitor rucaparib led to enhancedantitumor effects in vitro (59).

A phase Ib trial investigating the effects of olaparib combinedwith trabectedin in STS reported a PR in 18% and SD in 23% ofpatients (NCT02398058; Table 2; ref. 62). A phase I trial inpediatric and AYA solid tumors is investigating the efficacyof combined treatment of talazoparib with irinotecan, with orwithout temozolomide. Preliminary data encouraginglyshowed response in a subset of patients for the combinationof talazoparib and irinotecan (NCT02392793; Table 2; ref. 63).None of these studies have yet mentioned a response inrhabdomyosarcoma. One other trial is examining effects ofolaparib combined with concomitant radiotherapy in locallyadvanced STS (NCT02787642; Table 2).

Cell cycleIn addition to the DDR, the cell cycle can be inhibited to

affect cell viability. The cell cycle is a strictly regulated cellularprocess and multiple kinases are involved in its regulation,including the cyclin-dependent kinases (CDK), polo-like kinase1 (PLK1), and Wee1 kinase (64). CDKs play a crucial rolethroughout the whole cell cycle and act at different stages ofcell-cycle progression. PLK1 actively regulates the transitionfrom G2–M phase by phosphorylating Wee1, triggering Wee1degradation. Inhibition of PLK1 can induce a mitotic arrestleading to cell death. Wee1 negatively regulates entry intomitosis by inducing an inhibitory phosphorylation of CDK1,leading to a G2–M arrest necessary for DNA repair. Inhibition isthought to prevent the G2–M arrest and subsequent DNA repairresulting in a premature entry into mitosis and induction ofcell death (Fig. 1C).

PLK1. Rhabdomyosarcoma has higher levels of PLK1 comparedwith healthy tissue (65, 66). Multiple preclinical studies haveshown ARMS to be more sensitive to PLK1 inhibitors comparedwith ERMS (65–67). This sensitivitymight be related to the role ofPLK1 in the activation and expression of PAX3-FOXO1 (68). Allstudies showed synergistic effects for the combination of PLK1inhibitors and antimicrotubule agents (65–67). No synergismwas observed for the combination with etoposide, doxorubicin,or paclitaxel (65, 66). The working mechanism of etoposide,doxorubicin, and paclitaxel is either before (etoposide) or after(doxorubicin, paclitaxel) the mitotic phase, which might explainthe observed ineffectiveness of these combinations (66). In con-trast to PLK1-catalytic domain inhibitors, polo-box domain inhi-bitors in combination with vincristine were less effective;although it still induced apoptosis to a larger extend than respec-tive monotherapies (67). One phase I study with monotherapyvolasertib in pediatric solid tumors has been concluded(NCT01971476); however, results are not yet available. Despitethese promising preclinical results, there are no clinical trialsexamining combination treatments with PLK1 inhibitors in(pediatric) patients with rhabdomyosarcoma. However, basedon the preclinical data and the tolerable toxicity of the PLK1inhibitor NMS-1286937 in adult patients with solid tumor, suchtrials would be of interest (69).

Wee1. An in vitro drug screen identified compounds withpotential antitumor effects in rhabdomyosarcoma. The mono-therapeutic and combined effects of cyclophosphamide, irino-tecan, etoposide, dactinomycin, virorelbine, the Wee1 inhibitorAZD1775, the multi-RTK inhibitor cabozantinib, and the pro-tease inhibitor bortezomib were tested. The results showed that





Table

2.Clinical

trialsexam

iningtargeted

therap

y-based

combinationtrea

tmen

tsin

patientswith(softtissue

)sarcoma

Categ

ory

Target(s)

Combinationa

Pha

sePopulation

Response

Toxicity

NCT#

/Ref.

RTK

sIGF1R

IGF1R

þSFK

Gan

itum

abþ

dasatinib

I/II

ARMSan

dERMS

——

NCT030

41701

IGF1R

þmTOR

Cixutum

umab

þtemsirolim

usII

Bone

andSTSpatients,allocated

based

onIGF1R

expression(RMS

n¼

10)

PR:1/57IGF1Rþ

STSb

Grade3-4:8

%ofad

verseev

ents

(n¼

2546)

(13)

IGF1R

þmTOR

Cixutum

umab

þtemsirolim

usI

Ped

iatric

andAYArecurren

tsolid

tumors

(RMSn¼

9)

SD:3

/39(�

3cycles)No

men

tionofRMS.

Mostly

reve

rsible.D

ose-lim

iting

mucositis,fatigue

,hy

percholesterolemia,

tran

saminitisobserved

(14)

IGF1R

þmTOR

Cixutum

umab

þtemsirolim

usII

Ped

iatric

andad

olescen

tsarcoma

(RMSn¼

11)

SD:2

/11RMS

1RMSremove

ddue

totoxicity.

Others:ad

ditionoftemsirolim

usincrea

sedtoxicity

(15)

IGF1R

þmTOR

Dalotuzu

mab

þridaforolim

usI

Ped

iatric

advanced

solid

tumors

(RMSn¼

4)

PR:1/24Nomen

tionofRMS

Welltolerated

(16)

IGF1R

þchem

otherap

yCixutum

umab

or

temozo

lomideþ

combination

chem

otherap

y

IIPed

iatrican

dad

ultmetastaticARMS

andERMS

Cixutum

umab

vs.

temozolomide:

18-m

onths

EFS:

68%

and

39%,respective

ly

Cixutum

umab

:Grade5SOS:1/97

Grade4SOS:2

/97Te

mozolim

ide:

Notoxicity

men

tione

d

NCT01055

314/(17)

VEGF(R)

VEGFþ

chem

otherap

yBev

acizum

abþ

chem

otherap

yII

Childho

odan

dAYAmetastaticRMS

andno

n-RMSSTS

Che

motherap

yvs.

chem

otherap

yþ

bevacizum

ab:

Med

ianEFS(m

onths):

14.9

vs.2

0.6

OR:36

%vs.5

4%

Noincrea

segrade3/4toxicity

compared

tomono

chem

otherap

yNCT006435

65/(20)

VEGFþ

multi-RTKþ

chem

otherap

yBev

acizum

abþsorafenibþ

low-dose

cyclopho

spha

mide

IRecurrent/refractory

ped

iatric

and

YAsolid

tumors

(RMSn¼

2)PR:1/2RMS

Welltolerated

(21)

PDGFRa

Multikina

seþ

MEK

Pazopan

ibþ

tram

etinib

Ib/II

Advanced

STS(sinona

salE

RMSwith

PI3KCAE54

2Kab

erration

progressed

onPI3Kmono

therap

y,n¼

1)

PR:1/1

RMS,59%tumorsize

reduction

18/25:

grade1/2toxicity

3/25

:grade

3toxicity

1/25

:hea

rtproblemsdue

topriortrea

tmen

t

(32)

Downstrea

msigna

lingpathw

ays

mTO

RmTORþ

chem

otherap

yTe

msirolim

usþ

liposomal

doxo

rubicin

I/II

Recurrent

sarcoma(A

RMSn¼

2,ERMSn¼

2)-

Welltolerated

(40)

mTORþ

chem

otherap

yTe

msirolim

usþ

irinotecan

ortemozo

lomide

IYArelapsed/refractory

solid

tumors

(RMSn¼

4)

SD:1/4

RMS

Welltolerated

(41)

mTORþ

chem

otherap

ySirolim

usþ

vinb

lastine

IPed

iatric

recurren

t/refractory

solid

tumors

(RMSn¼

2)PR:1/2RMS(m

etastatic

ARMS)

Welltolerated

(42)

mTORþ

chem

otherap

ySirolim

usþ

cyclopho

spha

mideþ

topotecan

IPed

iatrican

dYArelapsed/refractory

solid

tumors

(RMSn¼

3)SD

:6/20Nomen

tionofR

MS

Welltolerated

(43)

Apoptosispathw

ayTR

AILR

TRAILR2þ

chem

otherap

yorIGF1R

Cona

tumum

abþ

ong

oing

chem

otherap

yor

gan

itum

ab

IIAdvanced

solid

tumors,including

sarcomas

––

NCT01327

612

(Continue

donthefollowingpag

e)

van Erp et al.




Table

2.Clinical

trialsexam

iningtargeted

therap

y-based

combinationtrea

tmen

tsin

patientswith(softtissue

)sarcoma

(Cont'd)

Categ

ory

Target(s)

Combinationa

Pha

sePopulation

Response

Toxicity

NCT#

/Ref.

DNAdam

ageresp

onse(D

DR)

PARP

PARP1þ

chem

otherap

yOlaparib

þTrabectedin

IMetastatican

dad

vanced

adult

sarcomas

PR:4

/22

SD:5

/22Nomen

tionRMS

Welltolerated

NCT023

98058

/(62)

PARP1þ

chem

otherap

yTa

lazo

parib

þirinotecan

with/witho

uttemozo

lomide

IPed

iatric

andAYArefractory

or

recurren

tsolid

tumors

Talazoparib

þirinotecan:

SD:9

/22Nomen

tionRMS

Welltolerated

NCT023

927

93/(63)

PARP1þ

radiotherap

yOlaparib

þco

ncomitan

tradiotherap

yI

Locally

advanced

/unresectable

STS

––

NCT027

876

42

Cellcycle

Wee1

Wee

1þ

chem

otherap

yAZD1775

þgem

citabine,

cisplatinorcarboplatin

IAdvanced

solid

tumors

PR:17/176

Confi

rmed

PR:7

/17

SD:9

4/176

Nomen

tionRMS

Welltolerated

NCT00648648/(72

)

Wee

1þ

chem

otherap

yAZD1775

þirinotecan

hydrochloride

I/II

Ped

iatric

andAYArelapsedan

drefractory

solid

tumors,including

RMS

––

NCT020

95132

CDK

CDK4/6

þchem

otherap

yRibociclib

þdoxo

rubicin

IAdultun

resectab

leSTS

––

NCT030

0920

1Epigen

ome

HDAC

HDACþ

VEGF

Valproic

acid

þbev

acizum

abþ

gem

citabineþ

doxetacel

I/II

Advanced

STS

––

NCT01106872

HDACþ

proteasome

Vorino

stat

þbortezomib

IIRecurrent

adultSTS

––

NCT00937

495

HDACþ

proteasome

Vorino

stat

þbortezomib

IPed

iatric

andad

olescen

tpatients

withrecurren

tan

drefractory

solid

tumors,includingsarcoma

––

NCT01132

911

HDACþ

chem

otherap

yVorino

stat

þetoposide

I/II

Childrenan

dad

olescen

tswith

refractory

solid

tumors,including

sarcoma

––

NCT01294670

HDACþ

chem

otherap

yAbex

inostat

þdoxo

rubicin

IMetastaticsarcoma

SD:5

/11Nomen

tionRMS

Wellm

anag

eable

(83)

HDACþ

chem

otherap

yBelinostat

þdoxo

rubicin

I/II

Advanced

STS(RMSn¼

1)Pha

seI:

PR:2

/25

SD:16/25

PD:7/25

Welltolerated

NCT00878

800/(84)

Pha

seII:

PR:1/16

CR:1/16

SD:9

/16

PD:5/16

Nomen

tionRMS

Abbreviations:þ,

IGF1R-positive

;—,n

otmen

tione

d;OR,o

bjectiveresponse;

PD,p

rogressivedisea

se;S

OS,sinusoidal

obstructivesynd

rome;

YA,y

oun

gad

ult.

aPub

chem

IDavailable

inSup

plemen

tary

Tab

leS1.

bSub

typeno

tspecified

.





for both rhabdomyosarcoma subtypes, AZD1775 combinedwith cabozantinib or bortezomib was most effective (70).In line with these findings, a multi-drug screen of pediatricorthotopic patient-derived xenograft (O-PDX) models alsoshowed high sensitivity of ARMS and ERMS for AZD1775.Moreover, the combination of AZD1775 with irinotecan andvincristine improved tumor response (71). In line with thepreclinical findings, the combined effects of AZD1775 andirinotecan hydrochloride will be examined in pediatric andAYA recurrent solid tumors, including rhabdomyosarcoma(NCT02095132; Table 2). In addition, AZD1775 combinedwith chemotherapy will be examined in adult solid tumorpatients (NCT00648648; Table 2; ref. 72).

CDK. CDK inhibitors could also be of interest for combinationtreatment in rhabdomyosarcoma. This could particularly be thecase in ARMS, because activation of cyclin D1/CDK4 leads toPAX3-FOXO1 activation. Preliminary data described synergismfor the combined treatment of CDK inhibitors with IGF1R orWee1 inhibitors in rhabdomyosarcoma cell lines (73, 74). Incontrast, combined treatment of the CDK4/6 inhibitor palboci-clib with doxorubicin showed antagonism in an ERMS cell line. Apossible biomarker for response could be expression of the cell-cycle–associated retinoblastoma protein (Rb). High Rb expres-sion correlated with a higher response to the combination ofpalbociclib with either doxorubicin or AZD1775, whereas knock-down of Rb led to antagonistic effects (74). One phase I studywillexamine the combined effects of the CDK4/6 inhibitor ribocicliband doxorubicin in adult unresectable STS and this might givemore insight into the clinical potential of this combination inrhabdomyosarcoma (NCT03009201; Table 2).

PAX3-FOXO1Compared with the abovementioned processes, targeting of

the PAX3-FOXO1 fusion in ARMS could lead to better resultswith a lower risk of treatment resistance. Direct targeting of thisfusion protein has, however, proven difficult, hence multiplestudies aimed at targeting pathways or proteins regulated bythis oncogenic driver. However, as the fusion orchestratesmultiple cellular processes and influences expression of manytarget genes, which are difficult to target simultaneously, robustantitumor responses in ARMS are difficult to achieve in thisway. In this light, direct silencing of the fusion with PAX3-FOXO1-silencing RNA (siRNA) could be a more effectiveapproach, as it would instantly deprive ARMS of its driver.Delivery of the siRNA to the tumor cell remains, however, anobstacle. A promising way of delivery could be a liposome-protamine-siRNA particle (LRP) that carries siRNA over thelipid barrier of the tumor cell. It has already been shown thatARMS can be targeted with LRPs when conjungated to cyclicRGD (arginine-glycine-aspartate) peptides, targeting the over-expressed aBb3 integrin receptor. RGD-LRP particles loadedwith anti-PAX3-FOXO1 siRNA (RGD-P3F-LRP) were effectivelydelivered to ARMS cell lines and led to reduction of bothPAX3-FOXO1 and PAX3-FOXO1-target gene expression. Someeffect on cell viability was seen, but no clear induction ofapoptosis. In vivo, tumor outgrowth was delayed comparedwith the control, but effects on established tumors were lim-ited, possibly due to the incomplete reduction of PAX3-FOXO1expression following treatment (75). Because no other treat-ments are currently capable of directly targeting this fusion

protein, the developments in nanomedicine are of interest,although the current data do not suggest fast implementationin the clinic. However, once the antitumor effects and bioavail-ability are optimized for clinical use, this could be a novel and,likely, highly effective treatment for patients with ARMS.

EpigenomeThe epigenome, alongside PAX3/7-FOXO1 gene transcription,

is responsible for the expression profile in rhabdomyosarcoma.Epigenetic changes affect chromatin, resulting in a more open ormore closed conformation. Open conformations allow for genetranscription while closed conformations repress gene expression(Fig. 1B). In tumors, epigenetic alterations lead to the repressionor induction of cancer-related genes. Several processes, includingDNA methylation and histone modification, are involved inepigenetic regulation (76).

Histone deacetylase. Histone deacetylases (HDAC) regulate thestructure of chromatin around histone proteins by deacetylat-ing lysine residues, leading to a compact structure. In manytumor types high levels of HDACs were shown to repress tumorsuppressor gene expression (76). In rhabdomyosarcoma,monotherapy with the HDAC inhibitor vorinostat had limitedeffects in vivo, which was similar to the effects observed in aphase I study in children with recurrent solid tumors. Never-theless, HDAC inhibitors combined with multiple chemother-apeutics were shown to have added effects in rhabdomyosar-coma cell lines (77–79). The combination with doxorubicinshowed the highest synergism and reduced rhabdomyosarcomatumor growth in vivo (78).

Dual and triple combinations, including vorinostat, wereexamined in ERMS. Multiple targeted therapies (vorinostat,17-DMAG, sorafenib, abacavir) were combined with eachother and/or with doxorubicin. Dual combinations of vorino-stat with the multi-RTK inhibitor sorafenib, and sorafenib withthe HSP90 inhibitor 17-DMAG were synergistic. In contrast,triple combinations with doxorubicin, 17-DMAG, and vorino-stat or sorafenib did not show a clear increase in antitumoreffects (80). One study examined multiple combinations in anARMS PDXmodel based on the genomic and proteomic profileof the tumor tissue, showing among others high Sirt1 (NADþ

HDAC) levels. In vivo combination treatments were designedand the combination of the HDAC inhibitor entinostat andthe chemotherapeutic docetaxel was most effective, with 65%tumor regression. Monotherapy entinostat led to a 43% tumorregression (81).

In addition to HDAC, lysine-specific demethylase 1 (LSD1)is overexpressed in rhabdomyosarcoma. Combination treat-ment of LSD1 and HDAC inhibitors showed synergism inARMS and ERMS cell lines (82). HDAC inhibitors in combi-nation with either targeted therapies or chemotherapy are inclinical trials (NCT01106872, NCT00937495, NCT01132911,NCT01294670; Table 2) and a subset of patients withadvanced STS responded to monotherapy and combinationtreatments (NCT00878800; Table 2; refs. 83, 84). Both thepreclinical and early clinical trial data show potential forepigenetic inhibitors combined with chemotherapy. It wouldbe interesting to see whether epigenetic inhibitors alter theexpression of repressed genes as this could lead to new com-bination treatments, possibly enhancing the antitumor effectsof epigenetic inhibitors.

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Epigenetic modification and immunotherapy. In line with thishypothesis, demethylating 5-aza-20-deoxytidine (DAC) enhancedthe expression of tumor antigens in rhabdomyosarcoma cell lines.DAC enhanced mRNA expression of immunogenic cancer-testisantigens MAGE-A1, MAGE-A3 and NY-ESO1. Of note, mRNAexpression did not necessarily correlate with protein expression,as only MAGE-A1 and –A3 were expressed in ARMS, whereasmRNA levels increased in both subtypes. The recognition-relatedproteins MHC class I–II molecules and costimulatory ICAM-1were also increased, as was the reactivity of cancer-testis antigen-specific CTLs. This indicated that, despite a low cancer testis-antigen increase, treatment of demethylating agents can enhancethe T-cell response against the tumor (85). No in vivo examinationhas been performed. However, these findings do suggest thattargeting of the epigenome can alter protein expression andmaketumor cells more vulnerable to other treatments, such asimmunotherapy.

DiscussionAs can be appreciated from the abovementioned studies, the

potential of combination therapy has been examined in manydifferent fields of targeted therapy. Recent studies show encour-aging data, suggesting that a variety of targeted therapy–basedcombinations are capable of increasing antitumor effects inrhabdomyosarcoma. The majority of these studies focused onthe potential of combinations with standard-of-care chemother-apeutics. In this regard, targeting of RTKs, downstream proteins,Hedgehog signaling, DDR, cell-cycle proteins and the epigenomeshows promise, especially as in many cases synergy was achievedwith low-dose chemotherapy, possibly lowering adverse events inpatients. Because chemotherapy is and will for the foreseeablefuture remain a vital part of rhabdomyosarcoma treatment, thesedrug combinations are of importance.

However, as chemotherapy-based treatments remain toxic,noncytotoxic-based targeted combination regimens have beenexplored as well, although to a lesser extent. Some combinationstargeting associated pathways, such as combined IGF1R andmTOR inhibition, show some efficacy in rhabdomyosarcoma.Simultaneous targeting of multiple cancer hallmarks that aretumor-specific and have complementary roles in tumor cellsurvival could hypothetically lead to more robust antitumoreffects with lesser adverse effects on healthy tissue. Indeed, com-binations targeting multiple distinct signaling pathways or dif-ferent cellular processes show potential for rhabdomyosarcomatreatment, including combined Hedgehog andmTOR inhibition,and HDAC inhibitors combined with multi-RTK inhibitors. Thecombination of certain epigenetic modifiers with immunomo-dulating drugs are also worthy of further assessment. More

research into this field is eagerly awaited. In addition to thetargeted therapies currently used in combination treatments,other targeted therapies, such as those directed against checkpointkinase 1 (CHK1) and enhancer of zeste homolog 2 (EZH2), couldbe of interest for future combination regimens. Preclinical datahave shown promising single-agent efficacy and combinationtreatments might be able to reduce treatment resistance and/orenhance the antitumor effects (86, 87).

This review also highlights some recent developments in nano-medicine and drug conjugates. Even though the current com-pounds are not yet ready for clinical implementation, these couldelicit high antitumor effects or even eliminate the ARMS driver.Further optimization of these compounds is therefore needed andexamination of their potential in rhabdomyosarcoma treatmentshould be continued.

Of note, most studies, both preclinical and clinical, showedtheir effects to be present in a subset of cell lines, xenografts orpatients, underlining the heterogeneous response these combi-nation treatments can generate. Discovery of predictive biomar-kers could decrease unnecessary treatment of patients and mightprevent unnecessary toxicities. Identification of biomarkers inrhabdomyosarcoma remains difficult, however, and moreresearch is necessary. Worthy of comment are the recent insightsfrom a variety of genomic, epigenomic, and (phospho)proteomicprojects. Although the majority of patients with rhabdomyosar-coma have a low mutational burden, a recent report did reveal anumber of activating mutations and epigenetic alterations thatcould have an effect on pathway activity (28). Also a phospho-proteomics screen in rhabdomyosarcoma cell lines, and a com-bined genomic and morphoproteomic screening of an ARMSsample identified and validated adequate treatment strategies(7, 81). This exemplifies that molecular genomics, epigenomics,and (phospho)proteomics might have a place in rhabdomyosar-coma diagnosis to provide patients with the best, most person-alized treatment available.

Altogether, a variety of targeted therapy–based combina-tion treatment regimens show promise for patients withrhabdomyosarcoma. Although more research is required intothe most suitable combinations accompanied with studiesidentifying predictive biomarkers, adequate implementationof such combined regimens in future clinical trials couldimprove outcome and reduce side effects of patients withrhabdomyosarcoma.

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

Received November 15, 2017; revised February 27, 2018; accepted May 1,2018; published first July 2, 2018.

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van Erp et al.



2018;17:1365-1380. Mol Cancer Ther Anke E.M. van Erp, Yvonne M.H. Versleijen-Jonkers, Winette T.A. van der Graaf, et al. Rhabdomyosarcoma

based Combination Treatment in−Targeted Therapy

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Targeted Therapy based Combination Treatment in ... · Targeted Therapy–based Combination Treatment in Rhabdomyosarcoma Anke E.M.van Erp1,Yvonne M.H.Versleijen-Jonkers1,Winette

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