preclinical characterization of novel chordoma cell ... ·...

Molecular and Cellular Pathobiology

Preclinical Characterization of Novel ChordomaCell Systems and Their Targeting byPharmocological Inhibitors of the CDK4/6Cell-Cycle PathwayAdrian vonWitzleben1, Lukas T. Goerttler1, Ralf Marienfeld1, Holger Barth2, Andr�e Lechel3,Kevin Mellert1, Michael B€ohm1, Marko Kornmann4, Regine Mayer-Steinacker5,Alexandra von Baer6, Markus Schultheiss6, Adrienne M. Flanagan7, Peter M€oller1,Silke Br€uderlein1, and Thomas F.E. Barth1

Abstract

Chordomas are tumors that arise at vertebral bodies and thebase of the skull. Although rare in incidence, they are deadlyowing to slow growth and a lack of effective therapeuticoptions. In this study, we addressed the need for chordomacell systems that can be used to identify therapeutic targetsand empower testing of candidate pharmacologic drugs. Eighthuman chordoma cell lines that we established exhibitedcytology, genomics, mRNA, and protein profiles that werecharacteristic of primary chordomas. Candidate responderprofiles were identified through an immunohistochemicalanalysis of a chordoma tissue bank of 43 patients. Genomic,mRNA, and protein expression analyses confirmed that the

new cell systems were highly representative of chordomatissues. Notably, all cells exhibited a loss of CDKN2A and p16,resulting in universal activation of the CDK4/6 and Rb pathways.Therefore, we investigated the CDK4/6 pathway and responses tothe CDK4/6–specific inhibitor palbociclib. In the newly validatedsystem, palbociclib treatment efficiently inhibited tumor cellgrowth in vitro and a drug responder versus nonresponder molec-ular signature was defined on the basis of immunohistochemicalexpression of CDK4/6/pRb (S780). Overall, our work offers avaluablenewtool for chordomastudies including thedevelopmentof novel biomarkers and molecular targeting strategies. CancerRes; 75(18); 3823–31. �2015 AACR.

IntroductionChordomas are rare tumors considered to arise from persis-

tent notochordal remnants along in the vertebral bodies (1).Because of their slow growth, there is no efficient standardchemotherapy: therapy of choice is surgery followed by radio-therapy. After surgical therapy of this orphan disease chordomarecurs in up to 50% of patients and metastasizes in up to 40%(1). Only a minority is cured by surgery, the disease-freesurvival is generally short (2–4). The median survival is 6 to7 years after diagnosis, although the range varies from months

to 25 years. High-dose radiotherapy is administered for residualor recurrent disease (5–10). Patients who have metastases andhave inoperable recurrent disease are not amenable for furtherradiotherapy. Therefore, there is a strong unmet need forsystemic pharmacologic therapy. Aggressive chemotherapy hasbeen described to be effective in rare cases of dedifferentiatedchordomas (11). Only limited response is seen by therapy withalkylating agents (ifosfamid), anthracyclines (doxorubicin),and cisplatin (12, 13). In the first prospective phase II studywith 9-nitrocamptothecin, one patient out of 15 achieved apartial remission (14). Recently, new targeted therapy optionsusing erlotinib, cetuximab, gefitinib, sunitinib, thalidomide,and lapatinib have been published (15–18). An activatedplatelet-derived growth factor receptor b (PDGFRb) wasdescribed as a basis for therapy with imatinib in patients withprogressive disease (19, 20).

Because of the rareness of this tumor and to only a few wellcharacterized chordoma cell lines there is limited experiencewith preclinical models (21–23). Lately, systematic pharmaco-logic screening of our first published two chordoma cell linesU-CH1 and U-CH2 has opened new insights for new potentialtargets (24).

Meanwhile, we have established and characterized 6 additionalchordoma cell lines. We found that the tumor suppressor p16(CDKN2A) is deleted and that p16 protein is not expressed inthese cell lines. This deletion has been described in themajority ofchordomas (25–27). The downstream machinery of p16 can beefficiently inhibited by CDK4/6 inhibitors such as palbociclib

1Institute of Pathology, University of Ulm, Ulm, Germany. 2Institute ofPharmacology and Toxicology, University of Ulm, Ulm, Germany.3Department of Internal Medicine I, University of Ulm, Ulm, Germany.4Department of General and Visceral Surgery, University of Ulm,Ulm, Germany. 5Department of Internal Medicine III, University of Ulm,Ulm, Germany. 6Department of Trauma Surgery, University of Ulm,Ulm, Germany. 7Cancer Institute, University College London (UCL),London, United Kingdom.

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

A. von Witzleben and L.T. Goerttler contributed equally to this article.

Corresponding Author: Peter M€oller, Institute of Pathology, M23, University ofUlm, Albert-Einstein-Allee 11, Ulm D-89081, Germany. Phone: 4907-31500-56320; Fax: 4907-31500-56384; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3270

�2015 American Association for Cancer Research.

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(28). Inhibition of this pathway by palbociclib is a definedmechanism to reduce the proliferation and growth of neoplasticcells (29, 30).

Because of the loss of p16 protein in chordoma cell lines, weinvestigated the downstreammolecules of this pathway in the celllines and in tissue comprising chordoma from 43 patients. Weanalyzed the effect of palbociclib on the growth of these cell linesand defined potential responder types.

Materials and MethodsCell culture and establishment of cell lines

The establishment of human chordoma cell lines U-CH1and U-CH2 was published by us in 2001 and 2010, respec-tively (31, 32). The new human chordoma cell lines (Nos.:U-CH3, U-CH6, U-CH7, U-CH10, U-CH11, U-CH12) were allestablished from sacrococcygeal chordomas using the sameprotocols (31, 32). The cell lines U-CH3, U-CH6, and U-CH7were derived from patients' tumors from the Royal NationalOrthopedic Hospital (Stanmore, Middlesex, United King-dom), and released from the Stanmore Musculoskeletal Bio-bank (Cambridgeshire 2 Research Ethics Committee; reference09/H0304/78). The cell lines U-CH1, U-CH2, U-CH10,U-CH11, and U-CH12 were derived from patients' tumorsfrom the Department of Trauma Surgery and the Departmentof General and Visceral Surgery, University of Ulm (Ulm,Germany). Approval for this procedure was obtained fromthe local ethics committee.

As controls for the Western blot analysis, we used the cell linesSK-LMS-1, CaCo2, and HEK-293; to control the effects of palbo-ciclib on cell proliferation in the MTS cell viability assay, MCF7and CAL51 cells served as references (Leibniz-Institut DSMZ;Bochum, Germany; ref. 33).

Comparative genomic hybridizationFor comparative genomic hybridization (CGH), theprotocol as

described by Lichter and colleagues (1995) was used. Imageacquisition was performed with the image analysis system ISIS(Meta-Systems).

FISH analysisFISH was performed according standard protocols using a

commercial available ZytoLight SPEC CDKN2A/CEN9 DUALCOLOR probe (Vysis/Abbott).

mRNA expression analysisAnalysis was performed according to the standard protocol

(Agilent Technologies) using the Whole Human Genome Micro-array (Design ID: 014850).

The generated data were analyzed using the program Gene-spring12 (protocol: http://www.chem.agilent.com/library/user-manuals/Public/G4140-90040_GeneExpression_OneColor_6.6.pdf, chip: http://www.genomics.agilent.com/en/Gene-Expres-sion-Human-Genome-Microarrays/Human-Gene-Expression-Microarrays/?cid¼AG-PT-131&tabId¼AG-PR-1078).

Western blot analysisThe following antibodies were used: brachyury (Santa Cruz

Biotechnology Inc., 1:1,000, Sc-20109), CDK4 (Zytomed Sys-tems, 1:1,000, 603-1840), CDK6 (Abcam, 1:1,000, Ab 54576),Rb (Cell Signaling Technology, 1:2,000, 9309), pRb (Ser780, Cell

Signaling Technology, 1:1,000, 9307), p16 (Santa Cruz Biotech-nology, 1:100, Sc-56330), cleaved caspase-3, and cleaved PARP(Cell Signaling Technology, 1:2,000, 9664 and 5625), b-actin(Sigma Aldrich, 1:1,000–1:10,000).

PalbociclibPalbociclib (PD-0332991) is a selective cyclin-dependent

kinase (CDK) inhibitor for CDK4 and CDK6, and inhibits thephosphorylation of the retinoblastoma protein (Rb). The reagentwas purchased as PD 0332991 isethionate from Sigma-AldrichInc.. Palbociclibwas dissolved inwater anddiluted in 10, 100, and1,000 nmol/L concentrations as published. For establishment ofthe IC50 values, palbociclib was used in concentrations of in 3, 6,10, 30, 60, 100, 300, 600, and 1,000 nmol/L.

AbemaciclibAbemaciclib (Ly2835219) is an alternative selective cyclin-

dependent kinase (CDK) inhibitor of CDK4 and CDK6 and waspurchased from Sellekchem. Abemaciclib was dissolved in waterand was used in concentrations of 3, 6, 10, 30, 60, 100, 300, 600,1,000, 10,000 nmol/L.

Flow cytometry (FACS)Cell lines U-CH1 and U-CH2 were subcultured and treated

with palbociclib (1,000 nmol/L) for 3 days. For cell-cycleanalysis, a protocol with propidium iodide (BD FACSCali-bur) and Cell Quest as detection software (BD Biosciences)were used. All FACS analysis experiments were performed astriplicates.

Cell countingAn inverse microscope coupled to a digital camera adapter

was used. The pictures were uploaded in the UTHSCSAImage Tool version 3.0 Final (http://compdent.uthscsa.edu/dig/itdesc.html). The control and the treated cell lines werecounted in 6 wells for calculation of the median with the SD.

Proliferation assayThe Alamar Blue Assay was used as outlined by the manufac-

turer using a 96-well plate reader (Spectramax 190).

Single tandem repeat analysisFor a genotyping of the cell lines U-CH1, U-CH2, U-CH3, U-

CH6, U-CH7, and U-CH12, we performed a single tandem repeat(STR) analysis using the PowerPlex 16 and Power Plex ESX 17 kit(Promega). For the cell lines U-CH10 and U-CH11 the Genome-Lab Human STR Primer Set Kit (Beckman Coulter) and theAmplitaq GOLD DNA Polymerase (Life Technologies) was used,respectively. The analysis was performed according to the man-ufacturers' handbook.

MTS cell proliferation assayCellTiter 96Aqueous One Solution Cell Proliferation Assay

from Promega was used to determine the cell viability. Therefore,the chordoma cell lines as well as MCF7 and CAL51 cell line werecultivated in 96-well plates and incubated with increasing con-centrations of palbociclib (0–1,000 nmol/L) in themedium.MTSwas added after 3 days of incubation at 37�C to the medium andthe absorption was measured at 490 nm using a microtiter platereader as described earlier (34).

von Witzleben et al.

Cancer Res; 75(18) September 15, 2015 Cancer Research3824




Chordoma tissue bankChordoma samples from 43 patients were available from the

Ulm tissue bank (median age: 69 years; range: 17–84 years; 26male, 17 female). Tumor size varied between 0.5 and 50 cm. Tenpatients had metastatic disease and 21 had recurrences. Thefollow-up data are available from 40 patients: 27 are alive (surveyrange from 0.5 to 234 months). The diagnosis was based onhistologic subtyping (WHO2013) and revealed inmost instancesa NOS (not otherwise specified) subtype, including chordomaswith focal chondroid, renal cell cancer like, and hepatoid differ-entiation (Supplementary Table S3).

Chordoma patient samples were pseudonymized to complywith theGerman law for correct usage of archival tissue for clinicalresearch (35). Approval for this procedure was obtained from thelocal ethics committee.

ImmunomorphologyImmunohistochemistry were performed on formalin-fixed,

paraffin-embedded tissue sections as described (36, 37). Theproportion of chordoma cells showing a positive immunoreac-tivity were categorized as follows: "no immunoreactivitydetected" (�); "immunoreactivity in up to 30%"(þ); immuno-reactivity in more than 30% and up to 70%" (þþ) and "immu-noreactivity in more than 70%" (þþþ) of the total number ofchordoma cells analyzed (Fig. 4A and B).

The following antibodies were used: polyclonal antibodyagainst S-100 protein (Dako, 1:1,000, Z0311), monoclonal anti-bodies against cytokeratin (AE1/AE3, Dako, 1:100, M3515),epithelial membrane antigen (EMA; E29, Dako, 1:500,M0613), vimentin (3b4, Dako, 1:300, M7020), brachyury (SantaCruz Biotechnology, 1:100, Sc-20109), CDK4 (Zytomed Systems,1:100, 603-1840), CDK6 (Abcam, 1:500, Ab 54576), Rb (CellSignaling Technology, 1:100, 9309), pRb (Ser780; Cell SignalingTechnology, 1:100, 9307), p16 (SantaCruzBiotechnology, 1:100,Sc-56330), Cyclin D1 (SP4, DCS, Cl677C01), and Ki-67 (cloneMIB-1; Dianova, 1:200, M7240).

Statistical analysisAll statistical tests were two-sided ANOVA and P values were

regarded as significant if they were lower than 0.05 (GraphPadPrism software, Version 6).

ResultsCharacterization of chordoma cell lines

We established 8 stable cell lines using standard protocols(Table 1, for typical cytology see Supplementary Fig. S2;

refs. 31, 32). All cell lines express the chordoma markerbrachyury (Supplementary Fig. S3). STR analyses were per-formed of the parental chordomas and the corresponding celllines in June 2015; these analyses confirmed the origin of thecell lines (Supplementary Table S5).

Additional information about these cell lines U-CH1, U-CH2,U-CH10, and U-CH11 can be requested via the ChordomaFoundation homepage (http://www.chordomafoundation.org/).

CGH data showed a highly overlapping pattern of recurrentaberrations and are thus regarded as representative onthe genomic level with recurrent gains on chromosome 7q(75%) and losses on chromosome 3 (75%), 9p and 10p(60% each; Supplementary Fig. S1; Supplementary TableS1). By FISH analysis, we found that all cell lines showedlosses of CDKN2A ranging from 50% to 86%, including abiallelic complete deletion of CDKN2A for U-CH10. Addi-tional FISH analyses revealed that this loss of CDKN2A wasalready present in the parental tumor of the chordoma celllines in a high percentage of cells analyzed (SupplementaryTable S2).

All cell lines showed high mRNA expression levels of typicalchordomas markers such as brachyury, collagen 2A1, and CD24(Supplementary Fig. S4A–S4C). The expression data sets wereclustered in an unsupervised approach [hierarchical clustering(nonaveraged) under Euclidean conditions in a centroid linkagerule] with the data of theNCI-60 panel of cancer cell lines. This is apublic available expression data set from the GEO database ofNCBI. These cell lines have been analyzed using a similar platformas for the chordoma cell lines. The chordoma cell lines (GEOaccession number: GSE68497) clustered together against thebackground of all cell lines.

We further included our own expression data of a nonchor-doma cartilaginous cell line (brachyury-negative), which featurescartilage differentiation and from tissue of a vertebral disk. Thesedata were analyzed with our chordoma cell lines data as well asexpression data from chondrocytes, liposarcomas, and pleomor-phic sarcoma cell lines. In this analysis, the chordoma cell linescluster together in one group and the cartilaginous cell line andthe sample from the vertebral tissue cluster together with chon-drocytes and the sarcoma cell lines; furthermore, the cluster of thechordoma cell lines intermingle with the data of the GEO expres-sion data and therefore support the cluster analyses (Supplemen-tary Fig. S5A and S5B).

A list of differentially expressed genes in correlation to the NCI-60 panel is available as Supplementary Table S4 (P < 10�4 andfold chance more than 10).

Table 1. Summarized data of the patients from which tissue the cell lines were established.

Clinical data Primary tumor Kinetics

Cell line LocalizationPrimary/

recurrences GenderAge at

diagnosis in years Size in cmHistologyprimary tumor

Tissueacquisition

Doublingtime in days

U-CH 1 Sacrum R M 45 20 NOS/renal 8/1998 2 dU-CH 2 Sacrum P F 70 13,6 NOS/renal 9/2000 4 dU-CH 3 Sacrum P F 65 11 NOS 6/2010 8 dU-CH 6 Sacrum Metastasis (skin) M Not known Not known NOS 11/2010 14 dU-CH 7 Sacrum P M 67 9 NOS 5/2011 7 dU-CH 10 Sacrum R F 74 13 NOS/renal 7/2000 14 dU-CH 11 Sacrum P M 71 8 NOS 9/2012 10 dU-CH 12 Sacrum P M 83 50 NOS 1/2013 7 d

NOTE: U-CH3, U-CH6, U-CH7 were established from tissue samples from A. Flanagan, UCL, London, United Kingdom.Abbreviation: NOS, Not otherwise specified.

Chordoma, Cell Lines, Palbociclib, Responder Types

www.aacrjournals.org Cancer Res; 75(18) September 15, 2015 3825




Analysis of the CDK4/6/phospho-Rb pathway in chordoma celllines

By FISH analyses, all chordoma cell lines and the parentaltumors of the cell lines we found a loss of CDKN2A in a highpercentage of cells (Supplementary Table S2). We conclude thatthis finding in the cell lines is not a secondary finding due to cellculture. We therefore focused our analysis on further componentsof the CDK4/6 pathway. In mRNA expression analysis, the cell-cycle molecules CDK4/6 and Rb are expressed on the mRNA level(data not shown) while we found a low mRNA expression ofCDKN2A. All analyzed cell lines are p16 protein negative, whileCDK4 and for CDK6 are protein positive (Fig. 1A).

Effect of palbociclib treatment on chordoma cell linesWe assumed that the CDK4/6 pathway is active in the cell lines

and performed cell growth inhibition experiments with palboci-clib. The pRb (S780) served as a marker molecule for the cellgrowth inhibition experiments.

After incubation of all cell lines with 100 nmol/L and 1,000nmol/L palbociclib for 3 days, they showed a strong decrease ofpRb (S780) expression with increase of palbociclib concentration(Fig. 1B). These data are supported by the immunocytologicalreactivity of U-CH1 with a reduction of the Ki-67 rating andreduction of pRb (S780; Supplementary Fig. S6). Prompted bythese results, we investigated the inhibitory effect of palbociclcibon the growth of the chordoma cell lines in more detail by

analyzing the amount of viable cells after palbociclib treatmentwith the MTS assay. Cells were incubated for 3 days withincreasing concentrations (0, 3, 6, 10, 30, 60, 100, 300, 600,1,000 nmol/L) of palbociclib and the amount of viable cellswas measured. For the analyzed cell lines U-CH2, U-CH3,U-CH6 and U-CH7, the IC50 value for palbociclib was between50 and 100 nmol/L (33), U-CH11 responded at higher con-centrations (IC50 between 300 and 400 nmol/L). In this test,MCF7 cells and CAL51 cells served as established positive andnegative control, respectively (38) (Fig. 3).

The inhibitory effect of palbociclib on the chordoma lines wasconfirmed by an alternative approach. The effect of a 6-daytreatment with 10, 100, and 1,000 nmol/L palbociclib on thegrowth of U-CH1 and U-CH7, U-CH10, and U-CH11 was ana-lyzed by cell counting (Fig. 2A). Moreover, the Alamar Blue assayconfirmed the concentration-dependent growth inhibition afterpalbociclib treatment of U-CH2, U-CH3, U-CH6, and U-CH10(Supplementary Fig. S7). All these lines responded to palbociclibwith an up to 18.5% decreased cell growth. Taken together, ourfindings demonstrate using different methods that palbociclibinhibits the growth of the 8 chordoma lines, however, the celllines showed different sensitivity toward palbociclib regardinggrowth inhibition.

The cell lines U-CH1 and U-CH2 were then used to analyze thecell cycle after treatment with 1,000 nmol/L of palbociclib for 3days. Figure 2B and C show the increase in the G1 phase anddecrease in S and G2 phase. To rule out increased apoptosis rates,we analyzed expression of cleaved PARP and cleaved caspase-3.We could not detect any cleaved PARP or cleaved caspase-3(Supplementary Fig. S8A and S8B), suggesting that in thesechordoma lines palbociclib inhibits cell proliferation rather thaninducing apoptosis.

In addition to palbociclib, we investigated the effects of analternative CDK4/6 inhibitor on the growth of the chordomacells and treated U-CH1 and U-CH6 cells with the establishedcompound LY2835219 (abemaciclib; ref. 39). We used thesetwo chordoma cell lines as these lines represent paradigmati-cally the chordoma cell lines regarding growth and morphol-ogy. The results indicated that treatment of these cells withLY2835219 also resulted in reduced cell viability (Supplemen-tary Fig. S9). Taken together, the results indicate that twodifferent pharmacologic inhibitors, which both interfere withCDK4/6 in cells in vitro, inhibited the growth of the chordomacell lines, implicating that this inhibition is due to the block ofthe CDK4/6 pathway.

Defining a potential responder versus potential nonresponderstatus

The protein expression profiles of the cell lines and the samplesobtained from the tissue bankwith respect to expression ofCDK4,CDK6, p16, Rb, pRb, Cyclin D1, and Ki-67 index were grouped todefinepotential responders. Basis for this scoringwas the responseto palbociclib of the chordoma cell lines (IC50 values and growthinhibition) as compared with the quantitative Western blot ratioof pRb (S780)/b-actin and a corresponding expression of pRb inthe chordoma samples. The potential responder type was strat-ified into groups (38, 40, 41). Every tumor tissue sample wasassessed for the expression of p16, CDK4, CDK6, Rb, pRb, CyclinD1, and Ki-67 index: 66% are negative for p16, 70% are positivefor CDK4, 85% are positive for CDK6, and over 90% are positivefor Rb and pRb (S780). Ki-67 index varied between 1% and 50%

Figure 1.A, Western blot analysis of 8 chordoma cell lines for p16, CDK4, and CDK6 ascompared with HeLa. B, inhibition assay of 8 chordoma cell lines for 3 dayswith 100 nmol/L and 1,000 nmol/L palbociclib. The first row shows theconcentration-dependent decrease of pRb expression; the second row givesthe Rb expression. b-Actin was used as loading control.






(Fig. 4B andC).On the basis of these results, we defined four typesof chordomas by scoring the expression profiles from 0 to 3(38, 40, 41). The potential nonresponder type (type 0) wasdefined by p16 expression in over 10% of the tumor cells. As Rband pRb are essential components of this pathway, lack of themolecules corresponds to a potential nonresponder. The absenceof both CDK4 and CDK6 protein is not compatible with thefunction of this pathway and therefore also classified as a poten-tial nonresponder status. The best assumed responder profile ischaracterized by expression of pRb (S780) in more than 70% ofcells (þþþ).

The other categories describe immunoreactivity for pRb(S780)in increasing percentages of positive cells: 0¼ p16 > 10%positive;Rb or pRb negative; CDK4 and CDK6 negative; 1¼ pRb isþ; 2¼pRb is þþ; 3 ¼ pRb is þþþ.

Applying the above categories to our cohort, we identified 5 of42 chordoma samples as potential nonresponders (type 0). Thepotential responder profiles of 41 samples were stratified asfollows, 10 (type 1), 13 (type 2), and 14 (type 3; Fig. 4A andB). Furthermore, on correlating the potential responder typeregarding the pRb expression with the Ki-67 index, we found asignificant positive correlation (P ¼ 0.0216), i.e., higher prolif-erating chordomas have a higher pRb expression. For the chor-doma cell lines, we found a trend correlating higher expression ofpRb with a higher Ki-67 index (P ¼ 0.08).

DiscussionIn the current study we characterized 6 newly generated

chordoma cell lines in direct comparison with two recentlydescribed chordoma lines. All these chordoma cell lines arenegative for p16 protein, most probable due to a loss of

CDKN2A in a high percentage of chordoma cells. In contrast,the downstream molecules CDK4/CDK6 and pRb are expressedin the cell lines. Inhibition of the chordoma cells leads to aconsistent reduction of growth when inhibited by the specificCDK4/CDK6 inhibitor palbociclib or an alternative CDK4/CDK6 inhibitor LY2835219 (abemaciclib). We show that ina cohort of chordoma tissue samples more than 80% of thepatients have a potential responder phenotype characterized byprotein expression of CDK4/6 and pRb.

Chordoma, being an orphan disease, is not treated uniformlywhen it comes to chemotherapy. However, due to screening onlarge scale by Xia and colleagues (24), there is increasing evidencethat chemotherapy targeting molecules like tyrosine kinases (e.g.,EGFR; refs. 15, 16, 42) may be an additional therapeutic option.The first xenograft experiments using the chordoma cell line U-CH1 showed that the cells preserve the typical cytology andimmunology, thus enabling further analysis of relevant pathwaysin situ (22).

ThemRNA expression profiling of our 8 cell lines revealed highmRNA levels of typical chordoma markers, including brachyury,collagen 2A1, and CD24 (32, 43). When compared with the NCI-60 cancer cell line panel and other public available mRNAexpression data the chordoma cell lines clustered together con-firming the characteristic profile of these tumors.

CDKN2A loss is a common genomic finding in chordoma (26,27). We found recurrent losses of CDKN2A in all chordoma celllines analyzed in a high percentage of cells. What is more, thisfinding was also present in the analyzed parental tumor, thereforeexcluding a secondary in vitro effect. The p16 protein wascompletely absent in all our chordoma cell lines.

As the tumor suppressor p16 (CDKN2A) plays a central role incontrolling the activity of CDK4 and CDK6 with respect to the cell

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Figure 2.A, cell counting graph of U-CH1after palbociclib inhibition for upto 6 days with 10, 100, and1,000 nmol/L. Green line, thecontrol cells. Other colors indicatethe increasing concentrations ofpalbociclib. B and C, FACS cell-cycle analysis forU-CH1 andU-CH2following 3 days of inhibition with1,000 nmol/L palbociclib. There isan increase of cells in G1 phase andreduction of cells in S and G2

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division cycle progression (26), a loss of p16 will lead to a tonicactivation of this pathway and thus is correlated negatively withprognosis, for example, in melanoma (44, 45). In line with thisobservation, the transcripts of CDK4 andCDK6 are upregulated onthe mRNA level in the chordoma cell lines, and CDK4 and CDK6proteins are consistently expressed in all our chordoma cell lines.

The next downstream molecule in the p16 pathway is the Rbprotein, which is phosphorylated by CDK4/6. In Western blotanalysis, all 8 chordoma cell lines expressed the Rb protein.

These cyclin-dependent kinases phosphorylate the Rb proteinat Serine 780 (33). The cell lines were all positive for pRb serine780. Taken together, the CDK4/6 pathway is active in all chordo-mas cell lines tested and shows important defects due to the loss ofthe negative regulator p16.

Palbociclib is a specific inhibitor of CDK4/6; correspond-ingly, the loss of p16 in lung cancer has been correlated with agrowth inhibitory effect of palbociclib while presence of p16

and loss of Rb is correlated with ineffectiveness to inhibition(40, 46). In this setting, we investigated whether inhibition ofthe p16 pathway would result in decreased pRb protein in thechordoma cell lines. We found that this protein was reduced ina dose-dependent manner after palbociclib treatment in thechordoma cell lines. In the inhibition experiments performedin the current study, the expression levels of pRb directlycorrelated with cell proliferation. In these experiments theIC50 values for four chordoma cell lines were below 100 nmol/Lof palbociclib and thus correspond to IC50 values reported forvarious other cancer cell lines (33). We further could show thatan alternative CDK4/CDK6 inhibitor (Ly2835219) has a sim-ilar inhibitory effect on the chordoma cell lines. Therefore, twobiochemically distinct inhibitors acting on CDK4/6 inhibitedthe growth of the chordoma cell lines, strongly suggesting thatthis inhibition is due to blocking the CDK4/6 pathway in thesecells (28).

Figure 3.Effect of palbociclib on the growth ofchordoma lines U-CH2, 3, 6, 7, and 11as well as Cal51. Cells were grown in96-well plates that were incubated for3 days at 37�C with increasingconcentrations of palbociclib (3, 6, 10,30, 60, 100, 300, 600, 1,000 nmol/L)in the medium or left untreated forcontrol. The amount of viable cellswasdetermined by using the MTS cellviability test (GraphPad Prismsoftware, Version 6).






We argued that growth inhibition of the chordoma cell wouldhave an effect on the cell cycle as the cascademight directly lead toa block at the G1 phase checkpoint. We show that this is the casewith a consequent increase of cells in G1 phase and a decrease of

cells in S and G2 phase. The sub-G1 phase was only slightlyincreased. These effects of palbociclib on the chordoma cells arenot due to increased apoptotic rates as shown by the absence of anincrease of cleaved caspase-3 or cleaved PARP. Therefore, we

Figure 4.Delineation of potential responder and nonresponder profiles to palbociclib treatment fromprotein expression data of the chordoma cell line in vitro as comparedwith theimmunohistologic expression of the variant constituents of the CDK4/CDK6 pathway in chordoma tissue in situ. A, chordoma cell line protein expression data indicatingan increase of growth inhibition from top to bottom and corresponding expression of elements of the CDK4/CDK6 pathway. B, expression of various elements ofthe CDK4/CDK6 pathway in chordoma tissue and assumed increasing probability to potential response to a palbociclib treatment; top, red, potential nonresponders;bottom, green, potential responders. Asterisk, chordoma sample no. 39. The corresponding morphology and immunohistochemistry is given in C. C,hematoxylin and eosin staining, immunohistochemistry, p16 negative, CDK6, CDK4, pRb, and Rb are strongly positive (from top to bottom); bars, 100 mm.






conclude that the consequence of palbociclib on the chordomacell lines is predominantly a growth inhibitory effect.

To dissect a potential responder phenotype from our tissuesample, we analyzed our tissue sample for the expression of p16,CDK4, CDK6, Rb, pRb, and cyclin D1 as compared with theresponding cell lines. By immunohistochemical typing, we strat-ified our patients' samples in various groups, leading to thedefinition of potential nonresponders and potential respondersto a treatment with palbociclib. The potential nonrespondergroup was defined as p16-positive, and absence of CDK4 orCDK6 or Rb/pRb. Vice versa, we established a potential responderphenotype characterized by the absence of p16 protein butdetectable CDK4/CDK6 and pRb (38, 40, 41).

More than 75% of chordoma samples were completelynegative for p16 pointing to a defect of this pathway in morethan two thirds of chordomas analyzed. Furthermore, immu-noreactivity for CDK4 and CDK6 in our chordoma tissuebanked samples showed that 56% of patients revealed coex-pression of CDK4 and CDK6, while 35% either expressed CDK4or CDK6, and only 5% were completely negative for thesemolecules. In addition, more than 90% of tissue samples wereRb- and pRb-positive.

Taken together, the analysis of more than 40 chordoma sam-ples led us to a ascribe chordoma patients to potential responder,who might benefit from a treatment with palbociclib. We showthat a higher Ki-67 index significantly correlated with higher pRbexpression. Therefore, these molecules are potential biomarkersfor tissue testing to identify a potential responder versus a poten-tial nonresponder status in chordoma tissue.

There is a number of clinical trials and studies with palbo-ciclib for various kinds of cancer such as squamous cellcarcinoma of head and neck, liver, ovarian, prostate, and breastcancer in phase II/III, melanoma in phase I–II, gastrointestinalstromal tumors, mantle cell lymphoma, and a phase II studyfor dedifferentiated liposarcoma, which all show favorableeffects on the progression-free survival and only moderate sideeffects (29, 41, 47–49). Our data strongly argue for an exten-sion of palbociclib therapy to chordoma patients with apotential responder phenotype as has been defined by ouranalysis. Therefore, the initiation of a prospective study includ-ing chordoma patients expressing the defined biomarkers ofthe CDK4/6 pathway by immunohistologic profiling to definea potential responder phenotype, as has been done for sub-

stances like tyrosine kinase inhibitors, has to be considered as apromising option for future therapy of chordomas in prospec-tive trials.

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

Authors' ContributionsConception and design: A. von Witzleben, R. Marienfeld, H. Barth,M. Schultheiss, P. M€oller, T.F.E. BarthDevelopment of methodology: A. von Witzleben, M. B€ohm, M. Schultheiss,P. M€oller, S. Br€uderlein, T.F.E. BarthAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. von Witzleben, L.T. Goerttler, H. Barth, A. Lechel,M. Kornmann, R. Mayer-Steinacker, A. von Baer, M. Schultheiss, A.M. Flanagan,P. M€oller, T.F.E. BarthAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. vonWitzleben, L.T. Goerttler, H. Barth, K. Mellert,M. Kornmann, P. M€oller, T.F.E. BarthWriting, review, and/or revision of the manuscript: A. von Witzleben,L.T. Goerttler, R. Marienfeld, K. Mellert, M. Kornmann, R. Mayer-Steinacker,A. von Baer, M. Schultheiss, A.M. Flanagan, P. M€oller, T.F.E. BarthAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. von Witzleben, P. M€oller, T.F.E. BarthStudy supervision: P. M€oller, S. Br€uderlein, T.F.E. Barth

AcknowledgmentsThe technical help of Jochen Lennerz,OlgaRitz, IwonaNerbas, JuliaMelzner,

Lena Kelsch, andMichaela Buck is greatly acknowledged. The authors thank theChordoma Foundation for financial support. The authors also thank thepatients for participating in the research and the clinicians and support staffof the London Sarcoma Service, United Kingdom.

Grant SupportCell lines U-CH3, U-CH6 and U-CH7 were derived from patients at the

RNOH and this component of the work was funded by Rosetrees Trust, UK.Support was provided to A.M. Flanagan (UCL) by the National Institute forHealth Research, UCLHBiomedical ResearchCentre, and theUCLExperimentalCancer Centre.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 5, 2014; revised May 4, 2015; accepted May 31, 2015;published OnlineFirst July 16, 2015.

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2015;75:3823-3831. Published OnlineFirst July 16, 2015.Cancer Res Adrian von Witzleben, Lukas T. Goerttler, Ralf Marienfeld, et al. Cell-Cycle PathwayTheir Targeting by Pharmocological Inhibitors of the CDK4/6 Preclinical Characterization of Novel Chordoma Cell Systems and

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