Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability

Author Manuscript Published OnlineFirst on December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0324 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title:

Afatinib is a new therapeutic approach in chordoma with a unique ability

to target EGFR and Brachyury

Paola Magnaghi1, Barbara Salom1, Liviana Cozzi1, Nadia Amboldi1, Dario Ballinari1, Elena

Tamborini2, Fabio Gasparri1, Alessia Montagnoli1, Laura Raddrizzani1, Alessio Somaschini1,

Roberta Bosotti1, Christian Orrenius1, Fabio Bozzi2, Silvana Pilotti2, Arturo Galvani1, Josh

Sommer3, Silvia Stacchiotti2 and Antonella Isacchi1

1 Oncology, Nerviano Medical Sciences, Nerviano (MI), Italy

2 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

3 Chordoma Foundation, Durham, NC USA

Running Title: Afatinib targets EGFR and brachyury in chordoma

Keywords: Chordoma, EGFR, afatinib, brachyury, clinical

Financial information: No financial support to declare Corresponding Author: Paola Magnaghi, Oncology, Nerviano Medical Sciences,

Nerviano (MI), Italy. Phone: + 39 0331581287; E-mail: [email protected]

Disclosure of Potential Conflicts of Interest

Silvia Stacchiotti is a Consultant/Advisory Board (Minor) member of Bayer. No potential conflicts of interest were disclosed by the other authors.

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0324 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Chordomas are rare bone tumors with no approved therapy. These tumors express several

activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine

kinase inhibitors. Although clinical benefit was observed in Phase II clinical trials with

imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to

date have shown modest activity. With the goal of identifying new drugs with immediate

therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRβ and EGFR tyrosine kinases, and assessed their anti-

proliferative activity against a panel of chordoma cell lines.

Chordoma cell lines were not responsive to MET and PDGFRβ inhibitors. U-CH1 and UM-

Chor1 were sensitive to all EGFR inhibitors, while the remaining cell lines were generally

insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the

chordoma panel. We then investigated the molecular mechanisms behind the responses

observed and found that the anti-proliferative IC50s correlate with the unique ability of

afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor

considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1,

SF8894, CF322 and CF365 chordoma tumor models in vivo. In the panel analyzed, high

EGFR phosphorylation and low AXL and STK33 expression correlated with higher

sensitivity to afatinib and deserve further investigation as potential biomarkers of response.

These data support the use of afatinib in clinical trials and provide the rationale for the

upcoming European phase II study on afatinib in advanced chordoma.

Introduction

Chordomas are primary malignant bone tumors that arise along the axial skeleton, usually in

the sacrum or skull-base, but also with low frequency in the mobile spine. Chordomas are

rare tumors, with an incidence below 1:1,000,000, and account for 1-4% of all primary bone

malignancies. They are typically late onset tumors with a peak incidence between the fifth

and sixth decade of life, but can also occur in children and young adults.

Chordomas are slow growing tumors, but are characterized by a high recurrence rate even

after complete surgical resection of the primary tumor. Distant metastasis occurs in 20-30%

of cases (1), but local recurrence affects >50% of patients (2). In case of relapse, surgery or

radiation therapy become challenging and patients usually die of their disease. Due to the

location of these tumors along the neuro-axis, patients commonly experience physical

dysfunctions and significant pain requiring morphine derivatives and steroids (1, 3). No

standard medical therapy is currently available, and chordomas are resistant to cytotoxic

chemotherapy.

Chordomas arise from embryonic notochordal remnants, and are characterized by expression

of the “T” gene product “brachyury”, a notochord-specific transcription factor essential for mesodermal specification and differentiation during development ( 4 ). The anomalous

expression of brachyury in adult notochordal remnants is believed to play a major role in the

onset and maintenance of chordoma (5, 6). Brachyury silencing in chordoma cell lines was

shown to impair cell proliferation and induce senescence, and attempts to target brachyury-

expressing cells through a vaccine are currently ongoing (7, 8, 9). Multiple studies have

shown that chordomas commonly exhibit expression and activation of tyrosine kinase

receptors and downstream signaling molecules, with MET, PDGFRβ and EGFR as the most

widely expressed, and HER2, KIT and VEGFR also expressed (10, 11, 12, 13, 14). 3

The availability of clinically approved drugs targeting EGFR and PDGFR has prompted the

evaluation of imatinib and lapatinib in phase II clinical trials for chordoma patients selected

for expression of corresponding drug targets (15, 16, 17, 18, 19). Imatinib demonstrated some

clinical benefit, although not achieving dimensional and long-lasting responses (16), while

lapatinib did not show a real clinical benefit (19). However, anecdotal responses to other

EGFR inhibitors have been reported, suggesting that EGFR inhibitors might have therapeutic

potential for these tumors (20, 21, 22, 23, 24). More recently, a phase II clinical trial with

sorafenib was conducted on chordomas, based on the in vitro activity of this drug on VEGFR

and PDGFR family members. In this study, patients were not selected or characterized for

expression of the corresponding receptors. Notably, sorafenib actually achieved a longer

progression free survival compared to imatinib (25).

Systematic preclinical studies to identify drugs active in chordomas have been limited in the

past by the paucity of biological models, as U-CH1 and U-CH2 cell lines represented the

only two available validated chordoma cell lines (26). More recently, a few additional bona fide chordoma cell lines have become available through the Chordoma Foundation, a patients advocacy organization (www.chordomafoundation.org).

With the aim of identifying kinase inhibitor drugs that might have therapeutic interest for

chordoma patients, we assembled a representative panel of chordoma cell lines and assessed

their sensitivity to EGFR, PDGFR and MET inhibitors, including approved drugs and other

representative compounds.

Materials and Methods

Cell lines and culture conditions

U-CH1, U-CH2, JHC7 and UM-Chor1 cell lines were obtained from Chordoma Foundation.

MUG-Chor1 and U-CH2 (ATCC) cell lines were purchased from ATCC. Chor-IN-1 cell line

was established in house from a surgical sample (27). Chordoma cell lines were cultured in

collagen-coated plates with IMDM / RPMI1640 4:1 Medium (Gibco BRL, Gaithersburg,

MD, USA) at 37°C in a humidified atmosphere containing 5% CO2. Media were

supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Euroclone). All cell lines

were authenticated by STR analysis (AmpFlSTR® Identifier® PCR Amplification Kit,

Applied Biosystems, Foster City, CA, USA), and checked for mycoplasma presence

(MycoAlert™ Mycoplasma Detection Kit, Lonza LT07) upon arrival and within six months from resuscitation.

Reagents and antibodies

The following antibodies were used: anti-brachyury (Santa Cruz sc-20109 rabbit polyclonal),

anti-EGFR (Santa Cruz sc-03 rabbit polyclonal); anti-PDGFR (Cell Signaling 4564 rabbit

monoclonal), anti-MET (Santa Cruz sc10 rabbit polyclonal); anti P-MET (Cell Signaling

7896 rabbit monoclonal); anti-P-STAT3 (Cell Signaling 9131 rabbit polyclonal); anti-P-AKT

(Cell Signaling 4060 rabbit monoclonal); anti-GAPDH (Santa Cruz 25778 rabbit polyclonal);

anti- P-Tyr (Millipore 05321 clone 4G10 mouse monoclonal); anti-P-MAPK (Cell Signaling

4377 rabbit monoclonal); anti-Caspase3 (Cell Signaling 9662 rabbit polyclonal); anti-LC3B

antibody (600-1384) from NB (Novus Biologicals).

HRP-conjugated secondary antibodies were used 1:10000 (Immunopure goat anti-mouse and

Immunopure Goat anti-rabbit from Thermo-Scientific). The detection was performed using

SuperSignal west Pico Chemiluminescent substrate from Thermo Scientific.

Compound acquisition and management

Gefitinib (Iressa), imatinib (Glivec; Gleevec), erlotinib (Tarceva), sunitinib (Sutent) and

Doxorubicine were synthesized in house. Crizotinib (PF-2341066), cabozantinib (BMS-

907351; XL-184) and crenolanib (CP-868596; ARO-002) were purchased from Selleck

Chemicals. Afatinib (Gilotrif) was purchased from LC Laboratories and MedChem Express.

Neratinib (HKI-272), dacomitinib, rociletinib (CO-1686), osimertinib (AZD-9291), lapatinib

(Tykerb) and PHA-665752 were purchased from MedChem Express.

Identity and purity of compounds was confirmed by LC-HRMS analysis and 1H-NMR.

Compounds were dissolved in 100% 10 mM DMSO solutions and stored under controlled atmosphere at -20°C. All compound working stocks were prepared by serial dilutions in

DMSO and then diluted 1:1000 into cell culture medium, to reach a 0.1% final DMSO concentration in all wells.

Assessment of anti-proliferative activity of the compounds

Cells were seeded at 3600 cells/well in 384-well white clear-bottom plates (Greiner) 24 h

before treatment. Compounds (6 dilution points in duplicate) were added and cells incubated

for additional 144 h. After cell lysis, cell viability was determined by quantifying the ATP

content (CellTiter-Glo assay-Promega and an Envision instrument-PerkinElmer). Inhibitory

activity (IC50) was calculated by comparing treated versus control data using a sigmoidal

fitting algorithm.

Assessment of the mechanism of action of the compounds

0.4x106 exponentially growing cells were seeded in 60 mm plates 48 h before treatment.

Compounds were added to the medium and plates incubated for the indicated times. Cells were then washed twice with ice cold PBS, and lysed in RIPA buffer (50mM Hepes pH 7.5,

150mM NaCl, 1% TritonX-100, 1% Sodium Deoxycholate, 0.1% SDS, 10mM EDTA,

Protease inhibitors Cocktail (Sigma P8340), Phosphatase inhibitors Cocktail I and II (Sigma

P2850 and P5725). Lysates were clarified by centrifugation for 10 min at 16,000xg, samples

(20 µg each) fractionated in 4-12% SDS-PAGE and transferred onto a Nitrocellulose

Membrane (Whatman-Protan 10401196). Immunoblots were performed with the described antibodies in TBS 1X (Biorad) with 5% milk and 0.1% Tween 20.

RT-qPCR analysis

RNA was extracted following the RNeasy Mini Kit (Qiagen) manufacturer’s procedures.

RNA yield and purity were evaluated by 260nm UV absorption and 260/280 and 260/230 ratios using Nanodrop 1000 (Thermo Scientific). RNA was reverse-transcribed using

TaqMan Reverse Transcription Reagents (Applied Biosystems/Life Technologies) and random hexamer priming. Each sample (10-12 ng cDNA) was assayed in duplicate 12.5 µl reactions by Real Time quantitative PCR (qPCR), on a ABI Prism 7900HT Applied

Biosystems Sequence Detector (Life Technologies) using SYBR Green technology, with reagents and materials from Applied Biosystems/Life Technologies (Power SYBR® Green

PCR Master Mix). Target-specific and endogenous reference control (GUSB, PPIA, 18S rRNA) primers (300 nM) are detailed in the table below. Relative quantification of expression levels was calculated following ΔΔCt method [Livak, Applied Biosystems

Sequence Detector User Bulletin 2, https://www2.appliedbiosystems.com]. All procedures were performed according to manufacturer’s instructions.

Primer sequences:

EGFR: fw 5'-AAGTCCCCCAGTGACTGCT-3', rev 5'-TGGCTTCGTCTCGGAATTT-3'

T-brachyury: fw 5'-GTATGAGCCTCGAATCCACAT-3', rev 5'-

GATAAGCAGTCACCGCTATGAA-3';

STK33: fw 5’-CGACTATAGCCAGCAGTGTGA-3’, rev 5’-

TCTCTTCTGAGCTTGCCAAA-3’;

AXL: fw 5’-CACCTCCCTGCAGCTTTC-3’, rev 5’-CTCCAGCCCAACATAGCC-3’;

GUSB: fw 5'-GCATCCAAAAGACGCACTTC-3', rev 5'-CACCCACCACCTACATCGAT-

3';

PPIA: fw 5'-CCCACCGTGTTCTTCGACAT-3', rev 5'-TTTCTGCTGTCTTTGGGACCTT-

3';

18S rRNA: fw 5'-AGCTGGAATTACCGCGG-3', rev 5'-

TGACGAAAAATAACAATACAGGACTC-3';

In vivo studies

Preclinical studies were conducted through the Chordoma Foundation Drug Screening

Pipeline at South Texas Accelerated Research Therapeutics (START) under International

Animal Care and Use Committee-approved protocols.

Antitumor activity was tested in:

• U-CH1: xenograft of the U-CH1 cell line (28)

• SF8894: PDX model (29)

• CF322: new chordoma PDX model generated from a clival recurrent tumor of a 42 year

old male

• CF365: new chordoma PDX model generated from a clival poorly differentiated

metastatic tumor of a 11 year old male

For all models, athymic nude mice (Charles River Labs) between 6-8 weeks of age were

implanted subcutaneously with tumor fragments from host animals. Once tumors reached

approximately 150-250 mm3, animals were matched by tumor volume (TV) and randomized

to control and treatment groups. All groups were dosed at 20 mg/Kg PO daily, U-CH1 and

SF9984 group for 28 days, CF322 and CF365 groups to end. Initial dosing began Day 0.

Animals were observed daily and weighed twice a week. TV and animal weight data were

collected electronically using a digital caliper and scale; tumor dimensions were converted to

volume using the formula TV (mm3) = width2 (mm2) x length (mm) x 0.52. Endpoints were a

mean control TV of approximately 1–2 cm3. Percent tumor growth inhibition values were calculated and reported for treatment versus control groups using initial and final tumor measurements. Statistical analysis was performed using a two-way analysis of variance

(ANOVA) followed by the Dunnett’s multiple comparisons test.

EGFR Immunoprecipitation

Cell were lysed in 1X Cell Lysis Buffer CLB (20mM TRIS pH 7.5, 150mM NaCl, 1mM

EDTA, 1mM EGTA, 1% Triton X-100, Protease and Phosphatase inhibitor cocktail-EZBlock

Protease Inhibitor Cocktail EDTA-free BV-K272, EZBlock Phosphatase Inhibitor Cocktail

II, BV-K275-1EAEZ, Block Phosphatase Inhibitor Cocktail III, BV-K276-1EA-Vinci-

Biochem).

Protein G Sepharose 4 fast flow (17-0618 GE Healthcare) was washed 4X with ice-cold PBS,

pre-loaded with anti-EGFR antibody (SantaCruz sc-03) in PBS/TritonX-100 and incubated

over-night with gentle rocking at 4°C.

Cell lysates were pre-cleared by rocking at 4°C for 20’ with Protein G Sepharose previously washed 4X with ice-cold PBS and 1X with CLB buffer;

Pre-loaded resin/antibodies were incubated at 4°C rocking for 3 h with the cell lysate.

Samples were transferred on cytospin filter (CytoSignal cat. C00-100), washed 3X with CLB

buffer, and eluted at 95°C in Laemmli-SDS buffer 2X.

Results

We assembled a panel of chordoma cell lines which included cells of sacral origin like the

widely used U-CH1 (28) and U-CH2 (26), the recently established MUG-Chor1 (30) and

JHC7 (8), and the first available clival cell line UM-Chor1 (31). In addition, we tested Chor-

IN-1, a new cell line derived in house from a sacral chordoma surgical sample, shown to

meet validation criteria for chordoma cell lines (27). All cell lines were first authenticated by

Short Tandem Repeat (STR) fingerprinting. The STR profile of U-CH2 cells obtained from

two sources differed at a single locus, suggesting the acquisition of little differences during

their propagation (Supplementary Table S1). Both clones were therefore tested in parallel.

Immunoblot analysis showed that all seven cell lines expressed the “T” gene product

brachyury, considered a defining marker of chordoma (Fig. 1).

MET and EGFR were shown to display similar expression levels across the different cell

lines, and HER2 was barely detectable. PDGFRβ expression was more heterogeneous,

ranging from very low (U-CH1), to high expression (U-CH2 and UM-Chor1). The

downstream signaling molecules AKT, MAPK and STAT3 were differentially activated

across the different cell lines, with no specific correlation to receptor expression (Fig. 1).

To evaluate the importance of these tyrosine kinase receptors for the growth of chordoma

cells, we assembled a broad panel of potent MET, PDGFR, and EGFR inhibitors, including

approved drugs as well as other reference inhibitors. The anti-proliferative activity of

compounds was determined after 144 h incubation, to allow two population doublings,

considering the slow growth rate of chordoma cells (>60 h doubling time). In parallel, the

inhibitors were profiled on reference cell lines: gastric adenocarcinoma MKN-45 with MET

amplification ( 32 ), NSCLC NCI-H1703 with PDGFR amplification ( 33 ), epidermoid

carcinoma A431 with EGFR amplification were used as positive controls, while ovarian

carcinoma A2780 cell line was used as general negative control, for which no target-

dependent activity was expected.

Crizotinib (34, 35) and cabozantinib (36), two approved MET inhibitor drugs, and the MET

selective inhibitor PHA-665752 (35, 37) did not display relevant anti-proliferative activity against chordoma cell lines, with IC50s higher than those measured for the A2780 control cell line (Supplementary Table S2A).

A similar result was observed with several PDGFR inhibitors, including the approved drugs

sunitinib and imatinib (35, 38, 39) as well as crenolanib (40) (Supplementary Table S2B).

We then tested the clinically approved EGFR inhibitor drugs erlotinib, gefitinib, afatinib, and

lapatinib, which possess different potency and selectivity within the EGFR/HER2 family (35,

41, 42, 43, 44). All compounds were active on U-CH1 and UM-Chor1 cell lines, although

with different potency (Table 1A). In particular, afatinib was very potent against both cell lines, with IC50s of 0.014 and 0.023 µM, respectively, while the other three drugs exhibited

IC50s in the 0.1-0.8 µM range. Moreover, afatinib was the only drug displaying activity, although with different potency, against all cell lines (IC50<0.7 µM), with the exception of

JHC7. The other EGFR inhibitors erlotinib, lapatinib and gefitinib, despite being active on U-

CH1 and UM-Chor1 in the sub-micromolar range, did not generally show relevant anti-

proliferative activity against the other chordoma cell lines.

To investigate whether these results were correlated with the potency and /or the covalent

mechanism of EGFR inhibition of afatinib, we also tested the covalent EGFR inhibitors

neratinib (35, 45 ) and dacomitinib ( 46 ). Both inhibited U-CH1 and UM-Chor1 in the

nanomolar range, but had poor activity on the other cell lines, with comparable or higher

IC50s than those observed for the A2780 control cell line (Table 1B).

Interestingly, dacomitinib displayed IC50 values comparable to afatinib in U-CH1 and UM-

Chor1, but was not generally active in the other cell lines, therefore indicating that the

activity of afatinib across the chordoma panel is not just related to its biochemical potency

against EGFR. Of note, the T790M EGFR mutant-selective covalent inhibitors osimertinib

(AZD-9291) (47) and rociletinib (48) were inactive or poorly active in all chordoma cell

lines, as expected in the absence of EGFR mutations (13, 27, www.chordomafoundation.org).

These data suggest that the anti-proliferative effect observed with afatinib against chordoma

is linked to EGFR inhibition, but is not just related to its potency or covalent mechanism of

action.

To obtain a dynamic view of the effects induced by treatment with afatinib, we performed a

kinetic live-cell analysis (see Supplementary Methods) using a multi-well automated

microscope (IncuCyte ZOOM). U-CH1 cells were treated with afatinib or doxorubicin

standard, and a time-lapse movie was generated integrating the sequence of live-cell images

acquired through 144 h treatment. Cell confluence was progressively decreased over time

with increasing concentrations of afatinib or doxorubicin, while increasing in the untreated

cells (Supplementary Fig. S1A). At active compounds concentrations, graphs bottomed to a background signal, representing the image analysis of cell debris. Conversely, caspase 3/7 induction (Supplementary Methods), measured with a specific fluorescent dye, was evident upon doxorubicin treatment, but was not detectable upon treatment with afatinib

(Supplementary Fig. S1B-C). This experiment demonstrated that afatinib inhibits U-CH1 cell proliferation and induces cell death in time- and dose-dependent manner, and indicated that the main mechanism of cell death is not apoptosis.

We then studied the dose- and time-dependent biomarker modulation following treatment with afatinib, erlotinib or lapatinib in U-CH1 cells, responsive to all EGFR inhibitors (Fig.

2). AKT and MAPK pathways were modulated in a dose-dependent manner by the three

inhibitors, both after short (2 h) and longer (48 h) incubation times, at doses consistent with anti-proliferative IC50s. STAT3 activation instead was not dependent upon EGFR signaling in these cells. Caspase 3 cleavage was not observed up to 48 h, again suggesting the involvement of mechanisms other than apoptosis in the anti-proliferative effect of these inhibitors (Fig. 2 and Supplementary Fig. S1B). Interestingly, treatment with afatinib and erlotinib induced a decrease in the total level of both the cytoplasmic and membrane-bound forms of LC3B, hinting a potential involvement of autophagic pathways.

A striking difference in the mechanism of action of the three compounds was the effect on brachyury and EGFR protein levels at the latest time points. After 48 h incubation, afatinib induced not only a strong decrease in the total level of EGFR, but also a dose-dependent decrease of brachyury protein. EGFR depletion was observed but only to a lesser extent upon treatment with erlotinib, and was absent after treatment with lapatinib. Brachyury expression was not affected by treatment with erlotinib or lapatinib.

To assess whether the afatinib–induced EGFR and brachyury down-modulation was linked to stronger potency or covalent mechanism of inhibition, we analyzed the effect of dacomitinib, that displayed similar IC50s on U-CH1 and UM-Chor1, but was generally inactive on the other cell lines (Table 1). Treatment of U-CH1 cells with dacomitinib induced a dose- dependent down-modulation of P-AKT levels, but, differently from afatinib, did not affect total levels of EGFR and brachyury up to 5 μM (Supplementary Fig. S2). These data support the hypothesis that the activity observed with afatinib against chordoma cell lines could be linked to its ability to ablate two proteins relevant for chordoma cells growth, and not merely to its potency or covalent mechanism of EGFR inhibition.

To investigate whether this particular mechanism occurred also in the other afatinib responsive chordoma cell lines, MUG-Chor1 and Chor-IN-1 were treated with the compound and analyzed by immunoblot. Fig. 3 shows that down modulation of EGFR and brachyury occurred in the same dose-response manner within each cell line, which correlated with the measured IC50s. Afatinib therefore turned out to be the only EGFR inhibitor displaying activity , although with different potency, across the chordoma cell line panel and the only inhibitor inducing a down-modulation of two biomarkers known to play a major role in chordoma cell growth.

To explore potential correlations between EGFR and brachyury, their reciprocal impact upon siRNA was analyzed (Supplementary Methods). Transfection of U-CH1 cells with specific siRNA oligonucleotides induced a complete ablation of the corresponding target protein

(EGFR or brachyury), with no effect on the other protein. In both cases, a strong impairment of cell viability was observed, therefore confirming that brachyury and EGFR play a crucial role in chordoma cell growth (Supplementary Fig. S3A-B). We ruled out that this down- regulation occurred transcriptionally, because treatment of U-CH1 cells with 0.1 and 1 μM afatinib for 24 or 48 h did not affect brachyury mRNA and only slightly decreased EGFR mRNA (Supplementary Fig. S4A).

Instead, treatment of U-CH1 cells with afatinib in the presence of MG-132 (proteasome inhibitor) or bafilomycin (autophagy inhibitor) prevented afatinib-induced EGFR and brachyury protein ablation (Supplementary Fig. S4B), therefore showing that the down- modulation of these proteins involves pathways of protein degradation.

We then assessed the activity of afatinib in vivo in U-CH1 xenografts and SF8894, CF322 and CF365 PDX (29) mouse models, generated as described in the methods section. Daily

oral treatment of mice with 20mg/Kg afatinib for the indicated times induced potent tumor

growth inhibition in all the analyzed models, with no apparent signs of toxicity (Fig. 4).

These data confirm that the activity observed in vitro with afatinib translates into impressive

in vivo efficacy in 4/4 models, providing a strong rationale for testing this drug in the clinic.

Finally, we exploited the cell lines with different levels of response to afatinib to start

exploring potential biomarkers of sensitivity to this drug. We investigated whether the

differences in the IC50s among these cell lines could be related to the total EGFR levels, to

the extent of receptor activation or to a differential response to the inhibitor. Total and

membrane-bound EGFR levels were found to be comparable among the different chordoma

cell lines, as detected by Flow Cytometry (FCM) analysis with cetuximab (Supplementary

Methods, Supplementary Fig. S5A).

The level of EGFR phosphorylation was analyzed, both in basal conditions and after

treatment with afatinib, by immuno-precipitation of the receptor from total cell lysates

followed by immunoblot with anti-P-Tyr antibody. Fig. 5A shows that EGFR is efficiently

immuno-precipitated from all cell lysates (lower panel), and that sensitive cell lines harbor

phosphorylated EGFR (upper panel), while JHC7 has a minimal level of basal

phosphorylation. Interestingly, treatment with afatinib induced complete EGFR de-

phosphorylation in U-CH1 cell line, but did not decrease the weak P-Tyr band detected in

JHC7 (Fig. 5A, upper panel). Since treatment with afatinib completely abolished EGFR

phosphorylation also in MUG-Chor1, Chor-IN-1 and U-CH2 cell lines (Supplementary Fig.

S5B), the band observed in JHC7 might be due to recognition of a different phosphorylation site, not linked to receptor activation. Therefore, the sensitivity of chordoma cell lines to afatinib seems to generally correlate with EGFR phosphorylation, and the lack of EGFR activation in JHC7 cell line likely accounts for the measured higher IC50.

To identify other potential determinants of differential sensitivity to afatinib, we analyzed the

expression of AXL, reported to mediate resistance to anti-EGFR therapies in different tumor

types (49). Fig. 5B shows that AXL expression inversely correlates with sensitivity to

afatinib, with a weak expression in U-CH1 and UM-Chor1 cell lines, and increased levels in

the less sensitive MUG-Chor1, U-CH2 and Chor-IN-1 cell lines. No expression of AXL was

observed in JHC7, where the EGFR pathway is not activated.

Interestingly, in a whole kinome-targeted sequencing, evaluating in parallel the differential

expression of ~500 kinases in U-CH1 versus the other sacral chordoma cell lines, STK33

emerged as the only kinase with undetectable expression in U-CH1, and higher expression in

the other cell lines (27). We then evaluated STK33 expression in all the cell lines by

RTqPCR and found no expression in both afatinib highly responsive U-CH1 and UM-Chor1 cell lines, while confirming relevant expression in the others.

Finally, we performed a pharmacodynamics study evaluating EGFR signaling as well as

AXL and STK33 levels after 1 and 2 h treatment of U-CH1, MUG-Chor1 and Chor-IN-1 cell

lines with afatinib. No relevant alterations of AXL and STK33 mRNA or total protein levels

were detected (Supplementary Fig. S6A-B). Interestingly, while EGFR phosphorylation was abrogated in all cell lines at 0.1 μM afatinib, P-AKT was shut-down in U-CH1 cell line, but

displayed a slight increasing trend in MUG-Chor1 and Chor-IN-1 (Supplementary Fig.

S6B-C), highlighting that the activity of afatinib in these cell lines is contributed by cellular

mechanisms that go beyond EGFR kinase inhibition.

Discussion

Only a few clinical trials have been conducted so far to assess the efficacy of targeted

therapeutic agents in chordoma (20, 21, 22, 23, 24), certainly limited by the exiguous number

of preclinical models of this indication available so far. We recently analyzed in depth at the

genomic level a panel of chordoma cell lines of sacral origin that included also the novel cell

line Chor-IN-1, established and characterized in our laboratories (27). With the goal of identifying new drugs that might have an immediate therapeutic application for chordoma patients, we evaluated the in vitro anti-proliferative activity of inhibitors of MET, PDGFR and EGFR tyrosine kinases, reported to be frequently expressed in chordomas.

MET inhibitors did not display significant activity and were not further investigated. PDGFR inhibitors were also not active, even in the cell lines with strong expression of PDGFRβ such

as U-CH2 and UM-Chor1. Considering that a phase II clinical trial with imatinib

demonstrated some clinical benefit for chordoma patients, PDGFRβ inhibition in the tumor

micro-environment might result in effects that go beyond mere anti-proliferative activity on

tumor cells in vitro, or PDGFRβ in tumors might be activated by paracrine mechanisms that

are lost in vitro. Interestingly, responses to imatinib observed in the clinic are primarily

related to changes in tumor features rather than dimension (17).

Multiple reports in the literature highlight the activity of different EGFR inhibitors in

chordoma cell lines, in animal models, and also sporadically in patients (reviewed in refs. 24,

50), providing evidence of a potential clinical relevance of EGFR inhibitors in chordoma, but

also raising questions about the most suitable agent.

We focused our studies on clinically approved EGFR inhibitors, and observed that all of them

displayed some activity, although with different potencies, against U-CH1 and UM-Chor1

cell lines. Afatinib was the most potent inhibitor in these cell lines, while lapatinib was the

least active, and was inactive on the remaining cell lines, which might have predicted the

poor activity observed in the clinic. Afatinib was also the only drug with activity across the

chordoma cell lines panel, with the exception of JHC7, that despite expressing significant

levels of EGFR is not driven by EGFR signaling.

The activity of afatinib in chordoma cell lines is particularly relevant because this drug is

very selective biochemically and its cellular activity is dependent upon EGFR pathway

activation (41, 42). The activity of afatinib in U-CH1 and UM-Chor1 chordoma cell lines is

equivalent to that reported for mutant EGFR-dependent lung tumors in vitro (42, 48), suggesting that chordoma might also represent a potential indication for this drug.

These cellular data were also corroborated by impressive in vivo efficacy, with tumor growth inhibition in one chordoma xenograft and in three different PDX models.

Scheipl et al. (51) published a focused compound screening describing the activity of EGFR

inhibitors in a subset of chordoma cell lines, reporting, in line with our data, that U-CH1 and

UM-Chor1 cell lines are sensitive to EGFR inhibitors, but unexpectedly they found afatinib

active on UM-Chor1 but not in U-CH1 cell line. Considering the potent activity we

repeatedly observed for afatinib on U-CH1 both in vitro, with modulation of downstream

pathways, and particularly in vivo, the lack of activity they observed in U-CH1 might be

related to differential screening conditions or compound handling.

Differently from erlotinib, gefitinib and lapatinib that are reversible inhibitors, afatinib

contains an electrophilic group capable of Michael addition to conserved cysteines within the

catalytic domains of EGFR (Cys797), HER2 (Cys805), and HER4 (Cys803), that induces

complete and long-lasting inhibition of receptor phosphorylation (42). However, the activity

of afatinib in chordoma cell lines appears to be related not only to its higher potency and

covalent mechanism, as neratinib and dacomitinib, sharing the same mechanism, were active

only in U-CH1 and UM-Chor1 cell lines.

This activity appears to be strongly contributed by its unique ability to down-modulate the

total level of EGFR and brachyury proteins, a feature not shared by the other inhibitors.

Interestingly, afatinib was recently shown to induce a down-modulation of the total level both

of HER2 and EGFR in tumors harvested from NCI-N87 xenografts treated with afatinib (52).

Brachyury is a distinctive marker of sporadic chordomas, duplication of the T gene locus is

typical of familial chordomas (6) and a DNA polymorphism in the T gene is associated with

chordoma predisposition in the general population (53). Accordingly, silencing of brachyury

in chordoma cell lines was widely shown to impair tumor growth both in vitro and in vivo (7,

8 and Supplementary Fig. S3B). Brachyury would represent an obvious target for drug development in chordomas, but direct inhibition of transcription factors with small molecules has thus far been extremely challenging.

The identification of a small molecule that, in addition to inhibiting EGFR signaling, induces

brachyury degradation represents a new approach indirectly targeting this important

transcription factor with a kinase inhibitor.

Overall, testing EGFR inhibitors across different cell lines and in vivo models suggests that a

subset of chordomas are driven by the EGFR signaling pathway and are strikingly sensitive

to EGFR inhibition. Pre-clinically this sensitivity correlates with the level of EGFR

phosphorylation and it will be important to assess if this holds through in the clinical study.

Interestingly, Mug-Chor, U-CH2 and Chor-IN-1 chordoma cell lines display a strong

expression of AXL receptor, which deserves further investigation, since it represents a

mechanism of resistance in lung cancer (49). Should this occur also in chordoma, a

combination therapy with AXL inhibitors, which are already in the clinic, might be beneficial

to these types of patients. STK33, which is absent in the afatinib most sensitive cell lines,

also represents a candidate biomarker for afatinib sensitivity which deserves further

investigation.

These data provide a strong rationale for formal evaluation of afatinib in the treatment of chordoma patients. Accordingly, a European phase II study on afatinib in advanced chordoma is about to start patient recruitment.

Acknowledgements

We acknowledge the Chordoma Foundation for providing U-CH1, U-CH2, JHC7 and UM-

Chor1 cell lines. We acknowledge the Johns Hopkins University for JHC7 and University of

Michigan for UM-Chor1 cell lines.

We acknowledge Clara Albanese, Stefano Camisasca, Antonella Landonio and Claudia Re from Nerviano Medical Sciences for technical support in Flow cytometry analysis and cell culturing, Michael Wick from START for technical support in the efficacy study.

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Table 1: Antiproliferative activity of EGFR inhibitors in chordoma cell lines.

IC50 (µM) (StdDev) UM- MUG- U-CH2 U-CH2 A431 A2780 Cell Line U-CH1 Chor-IN-1 JHC7 Chor1 Chor1 (Ch. F.) (ATCC) ctrl ctrl

Afatinib 0.014 0.023 0.258 0.494 0.531 0.668 1.346 0.026 1.915

(0.005) (0.007) (0.072) (0.409) (0.203) (0.351) (0.394) (0.009) (0.594)

Erlotinib 0.144 0.617 3.006 8.042 7.776 2.329 2.281 0.346 3.919

(0.049) (0.069) (0.977) (1.714) (1.953) (0.774) (0.848) (0.033) (0.898)

Lapatinib 0.656 0.516 >10 >10 >10 >10 >10 0.562 3.578

(0.257) (0.080) (-) (-) (-) (-) (-) (0.061) (0.771)

Gefitinib 0.791 0.751 6.241 6.259 5.936 9.040 7.010 0.333 4.762

(0.446) (0.055) (1.390) (2.502) (2.115) (1.389) (0.856) (0.069) (0.911)

B IC50 (µM) (StdDev) UM- MUG- U-CH2 U-CH2 Chor-IN- A431 A2780 Cell Line U-CH1 JHC7 Chor1 Chor1 (Ch. F.) (ATCC) 1 ctrl ctrl

Neratinib 0.038 0.110 1.946 1.926 3.253 2.009 2.893 0.282 0.603

(0.031) (0.023) (0.233) (0.189) (0.470) (0.569) (0.505) (0.059) (0.132)

Dacomitinib < 0.019 <0.019 2.075 2.400 2.145 0.418 1.230 0.043 2.715

(-) (-) (0.191) (0.042) (0.276) (0.054) (0.156) (0.014) (0.106)

Osimertinib 0.459 0.569 1.129 2.250 1.165 0.384 0.897 0.646 3.655 (T790M) (0.120) (0.034) (0.228) (0.085) (0.134) (0.021) (0.113) (0.199) (0.049)

Rociletinib 2.124 2.108 3.623 3.631 3.688 1.973 3.236 1.599 1.026 (T790M) (0.484) (0.586) (1.520) (1.152) (0.684) (1.250) (0.683) (1.527) (0.152)

Doxorubicin 0.166 0.067 0.455 0.152 0.348 0.340 0.881 0.083 0.010

(0.043) (0.040) (0.185) (0.038) (0.191) (0.119) (0.164) (0.004) (0.005)

In Bold: Registered EGFR inhibitors. The average IC50 (µM) where determined as described under materials and methods. All values were derived from technical duplicates and confirmed in multiple (n > 6) independent biological experiments.

The average IC50s (µM) were determined as described. All values represent technical duplicates and multiple (n > 6) independent experiments. A431 and A2780 cell lines are shown for comparison. Doxorubicin is used as reference standard. In Bold: registered drugs. A) Approved EGFR inhibitors B) Other advanced EGFR inhibitors

Figure Legends

Figure 1. Immunoblot analysis of tyrosine kinase receptors and other signaling

molecules in chordoma cell lines.

Immunoblot analysis of U-CH1, U-CH2, UM-Chor1, Chor-IN-1, MUG-Chor1 and JHC7

protein cell extracts, resolved on SDS-PAGE gel, transferred onto nitrocellulose membrane

and probed with the indicated antibodies.

Figure 2: Biomarker modulation upon treatment of U-CH1 cell line with different

EGFR inhibitors

Immunoblot analysis of U-CH1 cells treated with the indicated doses of inhibitors for 2 h

(upper panel) or 48 h (lower panel). Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with the indicated antibodies. IC50s of the different inhibitors are

reported.

Figure 3: Biomarker modulation upon treatment of U-CH1, MUG-Chor1 and Chor-IN-

1 cell lines with afatinib

Immunoblot analysis of U-CH1, MUG-Chor1 and Chor-IN-1 cells treated with afatinib for 2

h or 48 h. Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with

the indicated antibodies. IC50s for each cell line are reported.

Figure 4: In vivo efficacy of afatinib on U-CH1 xenograft and different PDX models

Nude mice bearing U-CH1 or SF8894, CF322 and CF365 tumors were dosed daily with

afatinib 20 mg/Kg po for 28 days (bar indicates the length of treatment) or to the end of

treatment, as indicated.

Figure 5: Identification of potential determinants of sensitivity to afatinib

A) Immuno-precipitation analysis of EGFR phosphorylation in the different chordoma cell lines. The receptor was immuno-precipitated with anti-EGFR antibody from untreated or afatinib-treated cell lines, as indicated. Immuno-complexes were resolved on SDS-PAGE gel.

Membranes were probed using an anti EGFR or anti-P-Tyr antibody.

B) Expression of AXL and STK33 in the different chordoma cell lines. Upper panel: immunoblot analysis of AXL expression. Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with anti-AXL antibody.

Lower panel: RTqPCR analysis of STK33 mRNA expression normalized to reference controls as described. Cell lines are ordered based on afatinib sensitivity ranking.

Figure 1

Brachyury

EGFR

PDGFR b

Met

Erb-b2

P-AKT (Ser473)

AKT

P-MAPK

MAPK

P-STAT3 (Thr705)

STAT3

GAPDH

Figure 2

C afatinib erlotinib lapatinib C Treatment (hours) 0.01 0.1 1 5 0.01 0.1 1 5 0.01 0.1 1 5 µM 2 p-AKT 48 (Ser473)

2 p-MAPK (Tyr202/Tyr204) 48

2 p-STAT3 (Thr705) 48

2 Casp. 3 48

8 LC3B 48

24 EGFR 48

8 Brachyury 48

48 GAPDH

IC50: 0.014 IC50: 0.144 IC50: 0.656 µM

Figure 3

U-CH1 MUG-Chor1 Chor-IN-1

Treatment C Afatinib C Afatinib C Afatinib (hours) 0.01 0.1 1 5 µM 0.01 0.1 1 5 µM 0.01 0.1 1 5 µM 2 p-AKT 48 (Ser473)

8 Brachyury 48

24 EGFR 48

48 GAPDH

IC50: 0.014 µM IC50: 0.258 µM IC50: 0.668 µM

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Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. mct.aacrjournals.org 20 mg/kg; 20 mg/kg; on September 26, 2021. © 2017American Association for Cancer Research. po; ; qdx28 po; qd to endto Author Manuscript Published OnlineFirst on December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0324 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5

+ + + : afatinib 1 µM 250 I.P. - EGFR tot. 150 W.B. -P-Tyr Afatinib sensitivity - +++ + + +++ + ++ 250 I.P. -EGFR tot. 150 W.B. -EGFR tot.

150 AXL 100

GAPDH

afatinib sensitivity

STK33 RT-qPCR Relative quantification Relative

Afatinib is a new therapeutic approach in chordoma with a unique ability to target EGFR and Brachyury

Paola Magnaghi, Barbara Salom, Liviana Cozzi, et al.

Mol Cancer Ther Published OnlineFirst December 13, 2017.

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