Afatinib Is a New Therapeutic Approach in Chordoma with A

Published OnlineFirst December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0324

Small Molecule Therapeutics Molecular Cancer Therapeutics 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

Abstract

Chordomas are rare bone tumors with no approved therapy. sitive to these drugs. Afatinib was the only EGFR inhibitor with These tumors express several activated tyrosine kinase receptors, activity across the chordoma panel. We then investigated the which prompted attempts to treat patients with tyrosine kinase molecular mechanisms behind the responses observed and found inhibitors. Although clinical beneﬁt was observed in phase II that the antiproliferative IC50s correlate with the unique ability of clinical trials with imatinib and sorafenib, and sporadically also afatinib to promote degradation of EGFR and brachyury, an with EGFR inhibitors, therapies evaluated to date have shown embryonic transcription factor considered a key driver of chor- modest activity. With the goal of identifying new drugs with doma. Afatinib displayed potent antitumor efﬁcacy in U-CH1, immediate therapeutic potential for chordoma patients, we col- SF8894, CF322, and CF365 chordoma tumor models in vivo.In lected clinically approved drugs and other advanced inhibitors of the panel analyzed, high EGFR phosphorylation and low AXL and MET, PDGFRb, and EGFR tyrosine kinases, and assessed their STK33 expression correlated with higher sensitivity to afatinib and antiproliferative activity against a panel of chordoma cell lines. deserve further investigation as potential biomarkers of response. Chordoma cell lines were not responsive to MET and PDGFRb These data support the use of afatinib in clinical trials and provide inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR the rationale for the upcoming European phase II study on afatinib inhibitors, whereas the remaining cell lines were generally insen- in advanced chordoma. Mol Cancer Ther; 17(3); 603–13. 2017 AACR.

Introduction patients usually die of their disease. Due to the location of these tumors along the neuro-axis, patients commonly experience Chordomas are primary malignant bone tumors that arise physical dysfunctions and significant pain requiring morphine along the axial skeleton, usually in the sacrum or skull-base, but derivatives and steroids (1, 3). No standard medical therapy is also with low frequency in the mobile spine. Chordomas are currently available, and chordomas are resistant to cytotoxic rare tumors, with an incidence below 1:1,000,000, and account chemotherapy. for 1% to 4% of all primary bone malignancies. They are Chordomas arise from embryonic notochordal remnants typically late onset tumors with a peak incidence between the and are characterized by expression of the "T" gene product fifthandsixthdecadesoflife,butcanalsooccurinchildrenand "brachyury", a notochord-specific transcription factor essential young adults. for mesodermal specification and differentiation during devel- Chordomas are slow growing tumors, but are characterized opment (4). The anomalous expression of brachyury in adult by a high recurrence rate even after complete surgical resection notochordal remnants is believed to play a major role in the onset of the primary tumor. Distant metastases occur in 20% to 30% and maintenance of chordoma (5, 6). Brachyury silencing in of cases (1), but local recurrences affect >50% of patients (2). In chordoma cell lines was shown to impair cell proliferation and case of relapse, surgery or radiotherapy become challenging and induce senescence, and attempts to target brachyury-expressing cells through a vaccine are currently ongoing (7–9). Multiple studies have shown that chordomas commonly exhibit expres- 1Oncology, Nerviano Medical Sciences, Nerviano, Milan, Italy. 2Fondazione IRCCS 3 sion and activation of tyrosine kinase receptors and downstream Istituto Nazionale dei Tumori, Milan, Italy. Chordoma Foundation, Durham, signaling molecules, with MET (HGF receptor), PDGFRb North Carolina. (PDGFRB), and EGFR as the most widely expressed, and HER2 Note: Supplementary data for this article are available at Molecular Cancer (ERBB2), KIT (SCFR), and VEGFR (KDR) also expressed (10–14). Therapeutics Online (http://mct.aacrjournals.org/). The availability of clinically approved drugs targeting EGFR Corresponding Author: Paola Magnaghi, Nerviano Medical Sciences, Viale L. and PDGFR has prompted the evaluation of imatinib and þ þ Pasteur 10, Nerviano, Milan, Italy. Phone: 390331581287; Fax: 390331581267; lapatinib in phase II clinical trials for chordoma patients E-mail: [email protected] selected for expression of corresponding drug targets (15– doi: 10.1158/1535-7163.MCT-17-0324 19). Imatinib demonstrated some clinical benefit, although 2017 American Association for Cancer Research. not achieving dimensional and long-lasting responses (16),

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whereas lapatinib did not show a real clinical benefit (19). Compound acquisition and management However, anecdotal responses to other EGFR inhibitors have Gefitinib (Iressa), imatinib (Glivec; Gleevec), erlotinib (Tar- been reported, suggesting that EGFR inhibitors might have ceva), sunitinib (Sutent), and doxorubicin were synthesized in therapeutic potential for these tumors (20–24). More recently, house. Crizotinib (PF-2341066), cabozantinib (BMS-907351; a phase II clinical trial with sorafenib was conducted on XL-184), and crenolanib (CP-868596; ARO-002) were purchased chordomas, based on the in vitro activity of this drug on VEGFR from Selleck Chemicals. Afatinib (Gilotrif) was purchased from and PDGFR family members. In this study, patients were not LC Laboratories and MedChem Express. Neratinib (HKI-272), selected or characterized for expression of the corresponding dacomitinib, rociletinib (CO-1686), osimertinib (AZD-9291), receptors. Notably, sorafenib actually achieved a longer pro- lapatinib (Tykerb), and PHA-665752 were purchased from Med- gression-free survival compared with imatinib (25). Chem Express. Systematic preclinical studies to identify drugs active in Identity and purity of compounds were confirmed by LC- chordomas have been limited in the past by the paucity HRMS analysis and 1H-NMR. Compounds were dissolved in of biological models, as U-CH1 and U-CH2 cell lines repre- 100% 10 mmol/L DMSO solutions and stored under controlled sented the only two available validated chordoma cell lines atmosphere at –20C. All compound working stocks were pre- (26). More recently, a few additional bona fide chordoma cell pared by serial dilutions in DMSO and then diluted 1:1,000 into lines have become available through the Chordoma Founda- cell culture medium, to reach a 0.1% final DMSO concentration in tion, a patients' advocacy organization (www.chordomafoun all wells. dation.org). With the aim of identifying kinase inhibitor drugs that might Assessment of antiproliferative activity of the compounds have therapeutic interest for chordoma patients, we assembled a Cells were seeded at 3,600 cells/well in 384-well white clear- representative panel of chordoma cell lines and assessed their bottom plates (Greiner) 24 hours before treatment. Compounds sensitivity to EGFR, PDGFR, and MET inhibitors, including (6 dilution points in duplicate) were added and cells incubated approved drugs and other representative compounds. for additional 144 hours. After cell lysis, cell viability was determined by quantifying the ATP content (CellTiter-Glo assay, Materials and Methods Promega using an Envision instrument, PerkinElmer). Inhibitory Cell lines and culture conditions activity (IC50) was calculated by comparing treated versus control U-CH1, U-CH2, JHC7, and UM-Chor1 cell lines were obtained data using a sigmoidal fitting algorithm. from Chordoma Foundation. MUG-Chor1 and U-CH2 (ATCC) cell lines were purchased from the ATCC. Chor-IN-1 cell line was Assessment of the mechanism of action of the compounds established in house from a surgical sample (27). Chordoma cell Exponentially growing cells (0.4 106) were seeded in 60 mm lines were cultured in collagen-coated plates with IMDM/ plates 48 hours before treatment. Compounds were added to RPMI1640 4:1 Medium (Gibco BRL) at 37 C in a humidified the medium and plates incubated for the indicated times. atmosphere containing 5% CO2. Media were supplemented with Cells were then washed twice with ice-cold PBS and lysed in 10% (v/v) heat-inactivated FBS (Euroclone). All cell lines were RIPA buffer (50 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 1% authenticated by short tandem repeat (STR) analysis (AmpFlSTR TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 10 mmol/L Identifier PCR Amplification Kit, Applied Biosystems) and EDTA, protease inhibitors Cocktail (Sigma; P8340), and phos- checked for mycoplasma presence (MycoAlert Mycoplasma phatase inhibitors Cocktail I and II (Sigma; P2850 and P5725). Detection Kit, Lonza LT07) upon arrival and within 6 months Lysates were clarified by centrifugation for 10 minutes at from resuscitation. 16,000 x g,samples(20mg each) fractionated in 4% to 12% SDS-PAGE, and transferred onto a Nitrocellulose Membrane Reagents and antibodies (Whatman-Protan; 10401196). Immunoblots were performed The following antibodies were used: anti-brachyury (Santa with the described antibodies in TBS 1X (Biorad) with 5% milk Cruz Biotechnology; sc-20109 rabbit polyclonal), anti-EGFR and 0.1% Tween 20. (Santa Cruz Biotechnology; sc-03 rabbit polyclonal), anti- PDGFRb (Cell Signaling Technology; 4564 rabbit monoclonal), RT-qPCR analysis anti-MET (Santa Cruz Biotechnology; sc10 rabbit polyclonal), RNA was extracted following the RNeasy Mini Kit (Qiagen) as anti P-MET (Cell Signaling Technology; 7896 rabbit monoclo- per the manufacturer's procedures. RNA yield and purity were nal), anti–P-STAT3 (Cell Signaling Technology; 9131 rabbit poly- evaluated by 260 nm UV absorption and 260/280 and 260/230 clonal), anti–P-AKT (Cell Signaling Technology; 4060 rabbit ratios using Nanodrop 1000 (Thermo Scientific). RNA was monoclonal), anti-GAPDH (Santa Cruz Biotechnology; 25778 reverse-transcribed using TaqMan Reverse Transcription Reagents rabbit polyclonal), anti–P-Tyr (Millipore; 05321 clone 4G10 (Applied Biosystems/Life Technologies) and random hexamer mouse monoclonal), anti–P-MAPK (Cell Signaling Technology; priming. Each sample (10–12 ng cDNA) was assayed in duplicate 4377 rabbit monoclonal), anti-Caspase3 (Cell Signaling Tech- 12.5 mL reactions by real-time qPCR, on a ABI Prism 7900HT nology; 9662 rabbit polyclonal), and anti-LC3B antibody (600- Applied Biosystems Sequence Detector (Life Technologies) using 1384) from NB (Novus Biologicals). SYBR Green technology, with reagents and materials from Horseradish peroxidase–conjugated secondary antibodies Applied Biosystems/Life Technologies (Power SYBR Green PCR were used 1:10,000 (Immunopure goat anti-mouse and Immu- Master Mix). Target-specific and endogenous reference control nopure Goat anti-rabbit from Thermo-Scientific). The detection (GUSB, PPIA, 18S rRNA) primers (300 nmol/L) are detailed in the was performed using SuperSignal West Pico Chemiluminescent table below. Relative quantification of expression levels was substrate from Thermo Scientific. calculated following the DDCt method (Livak; Applied Biosystems

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Afatinib Targets EGFR and Brachyury in Chordoma

Sequence Detector User Bulletin 2, https://www2.appliedbiosys Cell lysates were precleared by rocking at 4C for 20 minutes tems.com). All procedures were performed according to the with Protein G Sepharose previously washed 4X with ice-cold PBS manufacturer's instructions. and 1X with CLB buffer. Primer sequences: Preloaded resin/antibodies were incubated at 4C rocking for 3 EGFR: fw 5'-AAGTCCCCCAGTGACTGCT-3', rev 5'-TGGCTT- hours with the cell lysate. Samples were transferred on cytospin CGTCTCGGAATTT-3' filter (CytoSignal cat. C00-100), washed 3X with CLB buffer, and T-brachyury: fw 5'-GTATGAGCCTCGAATCCACAT-3', rev 5'- eluted at 95C in Laemmli-SDS buffer 2X. GATAAGCAGTCACCGCTATGAA-3'; STK33: fw 5'-CGACTATAGCCAGCAGTGTGA-3', rev 5'-TCTCT- Results TCTGAGCTTGCCAAA-3'; We assembled a panel of chordoma cell lines which included AXL: fw 5'-CACCTCCCTGCAGCTTTC-3', rev 5'-CTCCAGCC- cells of sacral origin like the widely used U-CH1 (28) and U-CH2 CAACATAGCC-3'; (26), the recently established MUG-Chor1 (30) and JHC7 (8), GUSB: fw 5'-GCATCCAAAAGACGCACTTC-3', rev 5'-CACC- and the first available clival cell line UM-Chor1 (31). In addition, CACCACCTACATCGAT-3'; we tested Chor-IN-1, a new cell line derived in house from a sacral PPIA: fw 5'-CCCACCGTGTTCTTCGACAT-3', rev 5'-TTTCT- chordoma surgical sample shown to meet validation criteria for GCTGTCTTTGGGACCTT-3'; chordoma cell lines (27). All cell lines were first authenticated by 18S rRNA: fw 5'-AGCTGGAATTACCGCGG-3', rev 5'-TGAC- STR fingerprinting. The STR profile of U-CH2 cells obtained from GAAAAATAACAATACAGGACTC-3'. two sources differed at a single locus, suggesting the acquisition of In vivo studies little differences during their propagation (Supplementary Table Preclinical studies were conducted through the Chordoma S1). Both clones were therefore tested in parallel. Foundation Drug Screening Pipeline at South Texas Accelerated Immunoblot analysis showed that all seven cell lines expressed fi Research Therapeutics (START) under International Animal Care the "T" gene product brachyury, considered a de ning marker of and Use Committee–approved protocols. chordoma (Fig. 1). 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. Brachyury

For all models, athymic nude mice (Charles River Labs) EGFR between 6 and 8 weeks of age were implanted subcutaneously with tumor fragments from host animals. Once tumors reached PDGFRb approximately 150 to 250 mm3, animals were matched by tumor volume (TV) and randomized to control and treatment groups. MET All groups were dosed at 20 mg/kg PO daily, U-CH1 and SF9984 group for 28 days, and CF322 and CF365 groups to end. Initial HER2 dosing began at day 0. Animals were observed daily and weighed twice a week. TV and animal weight data were collected electron- ically using a digital caliper and scale; tumor dimensions were P-AKT (Ser473) converted to volume using the formula TV (mm3) ¼ width2 (mm2) length (mm) 0.52. Endpoints were a mean control AKT TV of approximately 1 to 2 cm3. Percent tumor growth inhibition values were calculated and reported for treatment versus control P-MAPK groups using initial and ﬁnal tumor measurements. Statistical analysis was performed using a two-way ANOVA followed by the MAPK Dunnett multiple comparisons test. P-STAT3 (Thr705) EGFR immunoprecipitation Cell were lysed in 1X Cell Lysis Buffer CLB (20 mmol/L TRIS, STAT3 pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, Protease and Phosphatase inhibitor cocktail- GAPDH EZBlock Protease Inhibitor Cocktail EDTA-free BV-K272, EZBlock Phosphatase Inhibitor Cocktail II, BV-K275-1EAEZ, Block Phos- phatase Inhibitor Cocktail III, BV-K276-1EA-Vinci-Biochem). Figure 1. Immunoblot analysis of tyrosine kinase receptors and other signaling molecules Protein G Sepharose 4 fast ﬂow (17-0618; GE Healthcare) was in chordoma cell lines. Immunoblot analysis of U-CH1, U-CH2, UM-Chor1, washed 4X with ice-cold PBS, preloaded with anti-EGFR antibody Chor-IN-1, MUG-Chor1, and JHC7 protein cell extracts resolved on SDS-PAGE gel, (Santa Cruz Biotechnology sc-03) in PBS/TritonX-100 and incu- transferred onto nitrocellulose membrane, and probed with the indicated bated overnight with gentle rocking at 4 C. antibodies.

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MET and EGFR were shown to display similar expression levels exhibited IC50s in the 0.1–0.8 mmol/L range. Moreover, afatinib across the different cell lines, and HER2 was barely detectable. was the only drug displaying activity, although with different PDGFRb expression was more heterogeneous, ranging from very potency, against all cell lines (IC50 < 0.7 mmol/L), with the low (U-CH1) to high expression (U-CH2 and UM-Chor1). The exception of JHC7. The other EGFR inhibitors erlotinib, lapatinib, downstream signaling molecules AKT (PKB), MAPK (MAPK1/ and gefitinib, despite being active on U-CH1 and UM-Chor1 in MAPK3), and STAT3 were differentially activated across the different the submicromolar range, did not generally show considerable cell lines, with no specific correlation to receptor expression (Fig. 1). antiproliferative activity against the other chordoma cell lines. To evaluate the importance of these tyrosine kinase receptors To investigate whether these results were correlated with the for the growth of chordoma cells, we assembled a broad panel of potency and/or the covalent mechanism of EGFR inhibition of potent MET, PDGFRb, and EGFR inhibitors, including approved afatinib, we also tested the covalent EGFR inhibitors neratinib drugs as well as other reference inhibitors. The antiproliferative (35, 45) and dacomitinib (46). Both inhibited U-CH1 and UM- activity of compounds was determined after 144-hour incuba- Chor1 in the nanomolar range, but had poor activity on the other tion, to allow two population doublings, considering the slow cell lines, with comparable or higher IC50s than those observed for growth rate of chordoma cells (>60-hour doubling time). In the A2780 control cell line (Table 1B). parallel, the inhibitors were profiled on reference cell lines: gastric Interestingly, dacomitinib displayed IC50 values comparable adenocarcinoma MKN-45 with MET amplification (32), NSCLC with afatinib in U-CH1 and UM-Chor1, but was not generally NCI-H1703 with PDGFRA amplification (33), and epidermoid active in the other cell lines, therefore indicating that the activity of carcinoma A431 with EGFR amplification were used as positive afatinib across the chordoma panel is not just related to its controls, whereas ovarian carcinoma A2780 cell line was used as biochemical potency against EGFR. Of note, the T790M EGFR general negative control, for which no target-dependent activity mutant-selective covalent inhibitors osimertinib (AZD-9291; was expected. ref. 47) and rociletinib (48) were inactive or poorly active in all Crizotinib (34, 35) and cabozantinib (36), two approved MET chordoma cell lines, as expected in the absence of EGFR mutations inhibitor drugs, and the MET-selective inhibitor PHA-665752 (13, 27, www.chordomafoundation.org). (35, 37) did not display considerable antiproliferative activity These data suggest that the antiproliferative effect observed against chordoma cell lines, with IC50s higher than those mea- with afatinib against chordoma is linked to EGFR inhibition, but sured for the A2780 control cell line (Supplementary Table S2A). is not just related to its potency or covalent mechanism of action. A similar result was observed with several PDGFR inhibitors, To obtain a dynamic view of the effects induced by treatment including the approved drugs sunitinib and imatinib (35, 38, 39) with afatinib, we performed a kinetic live-cell analysis (see Sup- as well as crenolanib (ref. 40; Supplementary Table S2B). plementary Methods) using a multi-well automated microscope We then tested the clinically approved EGFR inhibitor drugs (IncuCyte ZOOM). U-CH1 cells were treated with afatinib or erlotinib, gefitinib, afatinib, and lapatinib, which possess differ- doxorubicin standard, and a time-lapse movie was generated ent potency and selectivity within the EGFR/HER2 family (35, 41– integrating the sequence of live-cell images acquired through 44). All compounds were active on U-CH1 and UM-Chor1 cell 144-hour treatment. Cell confluence was progressively decreased lines, although with different potency (Table 1A). In particular, over time with increasing concentrations of afatinib or doxoru- afatinib was very potent against both cell lines, with IC50s of 0.014 bicin, while increasing in the untreated cells (Supplementary and 0.023 mmol/L, respectively, whereas the other three drugs Fig. S1A). At active compound concentrations, graphs bottomed

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

IC50 (mmol/L; in brackets StdDev) Cell line U-CH1 UM-Chor1 MUG-Chor1 U-CH2 (Ch. F.) U-CH2 (ATCC) Chor-IN-1 JHC7 A431 ctrl A2780 ctrl A. Approved EGFR inhibitors 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. Other advanced EGFR inhibitors 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)

NOTE: In bold, registered EGFR inhibitors. The average IC50 (mmol/L) were determined as described under the Materials and Methods. All values were derived from technical duplicates and conﬁrmed in multiple (n > 6) independent biological experiments. A431 and A2780 cell lines are shown for comparison. Doxorubicin is used as reference standard.

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to a background signal, representing the image analysis of cell lapatinib in U-CH1 cells, responsive to all EGFR inhibitors debris. Conversely, caspase 3 (CASP-3) and caspase 7 (CASP-7) (Fig. 2). AKT and MAPK pathways were modulated in a dose- induction (Supplementary Methods), measured with a speciﬁc dependent manner by the three inhibitors, both after short ﬂuorescent dye, was evident upon doxorubicin treatment, but (2-8-24 hours) and longer (48 hours) incubation times, at was not detectable upon treatment with afatinib (Supplementary doses consistent with antiproliferative IC50s. STAT3 activation Fig. S1B and S1C). This experiment demonstrated that afatinib instead was not dependent upon EGFR signaling in these cells. inhibits U-CH1 cell proliferation and induces cell death in a time- Caspase 3 cleavage was not observed up to 48 hours, again and dose-dependent manner, and indicated that the main mech- suggesting the involvement of mechanisms other than apopto- anism of cell death is not apoptosis. sis in the antiproliferative effect of these inhibitors (Fig. 2; We then studied the dose- and time-dependent biomarker Supplementary Fig. S1B). Interestingly, treatment with afatinib modulation following treatment with afatinib, erlotinib, or and erlotinib induced a decrease in the total level of both the

C Afatinib Erlotinib Lapatinib C Treatment 0.01 0.1 1 5 0.01 0.1 1 5 0.01 0.1 1 5 m (hours) mol/L 2 p-AKT 48 (Ser473)

2 p-MAPK (Tyr202/Tyr204) 48

2 p-STAT3 (Thr705) 48

2 Figure 2. Biomarker modulation upon treatment of U-CH1 cell Casp. 3 line with different EGFR inhibitors. Immunoblot analysis of U-CH1 cells treated with the indicated doses of 48 inhibitors for 2-8-24 hours (top) or 48 hours (bottom). Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with the indicated antibodies. IC50s of the different inhibitors are reported. 8 LC3B 48

24 EGFR 48

8 Brachyury 48

48 GAPDH

IC50: 0.014 IC50: 0.144 IC50: 0.656 mmol/L

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cytoplasmic and membrane-bound forms of LC3B (MAP1LC3B), each cell line, which correlated with the measured IC50s. Afatinib hinting a potential involvement of autophagic pathways. therefore turned out to be the only EGFR inhibitor displaying A striking difference in the mechanism of action of the three activity, although with different potency, across the chordoma cell compounds was the effect on brachyury and EGFR protein levels line panel and the only inhibitor inducing a downmodulation of at the latest time points. After 48-hour incubation, afatinib two biomarkers known to play a major role in chordoma cell induced not only a strong decrease in the total level of EGFR, growth. but also a dose-dependent decrease of brachyury protein. EGFR To explore potential correlations between EGFR and brachyury, depletion was observed but only to a lesser extent upon treatment their reciprocal impact upon siRNA was analyzed (Supplementary with erlotinib, and was absent after treatment with lapatinib. Methods). Transfection of U-CH1 cells with specific siRNA oli- Brachyury expression was not affected by treatment with erlotinib gonucleotides induced a complete ablation of the corresponding or lapatinib. target protein (EGFR or brachyury), with no effect on the other To assess whether the afatinib-induced EGFR and brachyury protein. In both cases, a strong impairment of cell viability was downmodulation was linked to stronger potency or covalent observed, therefore confirming that brachyury and EGFR play a mechanism of inhibition, we analyzed the effect of dacomitinib, crucial role in chordoma cell growth (Supplementary Fig. S3A and which displayed similar IC50s on U-CH1 and UM-Chor1, but was S3B). We ruled out that this downregulation occurred transcrip- generally inactive on the other cell lines (Table 1). Treatment of U- tionally, because treatment of U-CH1 cells with 0.1 and 1 mmol/L CH1 cells with dacomitinib induced a dose-dependent down- afatinib for 24 or 48 hours did not affect brachyury mRNA and modulation of P-AKT levels, but, differently from afatinib, did not only slightly decreased EGFR mRNA (Supplementary Fig. S4A). affect total levels of EGFR and brachyury up to 5 mmol/L (Sup- Instead, treatment of U-CH1 cells with afatinib in the presence plementary Fig. S2). These data support the hypothesis that the of MG-132 (proteasome inhibitor) or bafilomycin (autophagy activity observed with afatinib against chordoma cell lines could inhibitor) prevented afatinib-induced EGFR and brachyury pro- be linked to its ability to ablate two proteins relevant for chor- tein ablation (Supplementary Fig. S4B), therefore showing that doma cells growth, and not merely to its potency or covalent the downmodulation of these proteins involves pathways of mechanism of EGFR inhibition. protein degradation. To investigate whether this particular mechanism occurred also We then assessed the activity of afatinib in vivo in U-CH1 in the other afatinib-responsive chordoma cell lines, MUG-Chor1 xenografts and SF8894, CF322, and CF365 PDX (29) mouse and Chor-IN-1 were treated with the compound and analyzed by models, generated as described in the Materials and Methods immunoblot. Figure 3 shows that downmodulation of EGFR and section. Daily oral treatment of mice with 20 mg/kg afatinib for brachyury occurred in the same dose-response manner within the indicated times induced potent tumor growth inhibition in all U-CH1 MUG-Chor1 Chor-IN-1

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

8 Brachyury 48

24 EGFR 48

48 GAPDH

m m m IC50: 0.014 mol/L IC50: 0.258 mol/L IC50: 0.668 mol/L

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 2-8-24 or 48 hours. Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with the indicated antibodies.

IC50s for each cell line are reported.

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the analyzed models, with no apparent signs of toxicity (Fig. 4). MUG-Chor1, Chor-IN-1, and U-CH2 cell lines (Supplementary These data confirm that the activity observed in vitro with afatinib Fig. S5B), the band observed in JHC7 might be due to recognition translates into impressive in vivo efficacy in four out of four models, of a different phosphorylation site, not linked to receptor activa- providing a strong rationale for testing this drug in the clinic. tion. Therefore, the sensitivity of chordoma cell lines to afatinib Finally, we exploited the cell lines with different levels of seems to generally correlate with EGFR phosphorylation, and the response to afatinib to start exploring potential biomarkers of lack of EGFR activation in JHC7 cell line likely accounts for the sensitivity to this drug. We investigated whether the differences measured higher IC50. in the IC50s among these cell lines could be related to the To identify other potential determinants of differential sen- total EGFR levels, to the extent of receptor activation or to a sitivity to afatinib, we analyzed the expression of AXL, reported differential response to the inhibitor. Total and membrane- to mediate resistance to anti-EGFR therapies in different tumor bound EGFR levels were found to be comparable among the types (49). Figure 5B shows that AXL expression inversely different chordoma cell lines, as detected by flow cytometry correlates with sensitivity to afatinib, with a weak expression analysis with cetuximab (Supplementary Methods; Supplemen- in U-CH1 and UM-Chor1 cell lines, and higher levels in the tary Fig. S5A). less sensitive MUG-Chor1, U-CH2, and Chor-IN-1 cell lines. No The level of EGFR phosphorylation was analyzed, both in basal expression of AXL was observed in JHC7, where the EGFR conditions and after treatment with afatinib, by immunoprecip- pathway is not activated. itation of the receptor from total cell lysates followed by immu- Interestingly, in a whole kinome-targeted sequencing, evaluat- noblot with anti–P-Tyr antibody. Figure 5A shows that EGFR is ing in parallel the differential expression of approximately 500 efficiently immunoprecipitated from all cell lysates (bottom) and kinases in U-CH1 versus the other sacral chordoma cell lines, that sensitive cell lines harbor phosphorylated EGFR (top), where- STK33 emerged as the only kinase with undetectable expression in as JHC7 has a minimal level of basal phosphorylation. Interest- U-CH1 and higher expression in the other cell lines (27). We then ingly, treatment with afatinib induced complete EGFR dephos- evaluated STK33 expression in all the cell lines by RT-qPCR and phorylation in U-CH1 cell line, but did not decrease the weak P- found no expression in both afatinib highly responsive U-CH1 Tyr band detected in JHC7 (Fig. 5A, top). Because treatment with and UM-Chor1 cell lines, while confirming relevant expression in afatinib completely abolished EGFR phosphorylation also in the others. 20 mg/kg; po; qdx28

UCH1 SF8894 2,000 1,200 1,800

1,600 1,000 )

3 1,400 800 1,200 Control 1,000 600

800 Afatinib 400 600 Tumor volume (mm Tumor 400 200 200 Figure 4. In vivo efﬁcacy of afatinib on U-CH1 0 0 0 10 20 30 40 50 0 10 20 30 40 50 xenograft and different PDX models. Days Nude mice bearing U-CH1 or SF8894, Days CF322, and CF365 tumors were dosed daily with afatinib 20 mg/kg po for 28 20 mg/kg; po; qd to end days (bar indicates the length of CF322 CF365 treatment) or to the end of treatment, 900 2,500 as indicated. 800

700 2,000 ) 3 600 1,500 500 Control

400 Afatinib 1,000 300 Tumor volume (mm Tumor 200 500 100

0 0 0 10 20 30 40 50 0 20 40 60 80 Days Days

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+ + + : Afatinib 1 mmol/L 250 I.P. a-EGFR tot. 150 W.B. a-P-Tyr Afatinib sensitivity - +++ + + +++ + ++ 250 I.P. a-EGFR tot. 150 W.B. a-EGFR tot.

150 AXL 100

GAPDH

Afatinib sensitivity 4 3.5 STK33 RT-qPCR 3 2.5 2 1.5 1 0.5 Relative quantification Relative 0

JHC7 U-CH1 UM-Chor1 Chor-IN-1 MUG-Chor1U-CH2 (CF) Pos controlNeg controlRef control U-CH2 (ATCC)

Figure 5. Identiﬁcation of potential determinants of sensitivity to afatinib. A, Immunoprecipitation analysis of EGFR phosphorylation in the different chordoma cell lines. The receptor was immunoprecipitated with anti-EGFR antibody from untreated or afatinib-treated cell lines, as indicated. Immunocomplexes 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. Top: immunoblot analysis of AXL expression. Protein cell extracts were resolved on SDS-PAGE gel and membranes probed with anti-AXL antibody. Bottom: RT-qPCR analysis of STK33 mRNA expression normalized to reference controls as described. Cell lines are ordered based on afatinib sensitivity ranking.

Finally, we performed a pharmacodynamics study evaluating certainly limited by the exiguous number of preclinical models of EGFR signaling as well as AXL and STK33 levels after 1 and 2 hours this indication available so far. We recently analyzed in depth at of treatment of U-CH1, MUG-Chor1, and Chor-IN-1 cell lines the genomic level a panel of chordoma cell lines of sacral origin with afatinib. No relevant alterations of AXL and STK33 mRNA or that included also the novel cell line Chor-IN-1, established and total protein levels were detected (Supplementary Fig. S6A and characterized in our laboratories (27). With the goal of identifying S6B). Interestingly, although EGFR phosphorylation was abro- new drugs that might have an immediate therapeutic application gated in all cell lines at 0.1 mmol/L afatinib, P-AKT was shut for chordoma patients, we evaluated the in vitro antiproliferative down in U-CH1 cell line, but displayed a slight increasing trend in activity of inhibitors of MET, PDGFRb, and EGFR tyrosine kinases, MUG-Chor1 and Chor-IN-1 (Supplementary Fig. S6B and S6C), reported to be frequently expressed in chordomas. highlighting that the activity of afatinib in these cell lines is MET inhibitors did not display significant activity and were not contributed by cellular mechanisms that go beyond EGFR kinase further investigated. PDGFR inhibitors were also not active, even inhibition. in the cell lines with strong expression of PDGFRb such as U-CH2 and UM-Chor1. Considering that a phase II clinical trial with imatinib demonstrated some clinical benefit for chordoma Discussion patients, PDGFRb inhibition in the tumor microenvironment Only a few clinical trials have been conducted so far to assess might result in effects that go beyond mere antiproliferative the efficacy of targeted therapeutic agents in chordoma (20–24), activity on tumor cells in vitro, or PDGFRb in tumors might be

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activated by paracrine mechanisms that are lost in vitro. Interest- Supplementary Fig. S3B). Brachyury would represent an obvious ingly, responses to imatinib observed in the clinic are primarily target for drug development in chordomas, but direct inhibition related to changes in tumor features rather than dimension (17). of transcription factors with small molecules has thus far been Multiple reports in the literature highlight the activity of dif- extremely challenging. ferent EGFR inhibitors in chordoma cell lines, in animal models, The identification of a small molecule that, in addition to and also sporadically in patients (reviewed in refs. 24, 50), inhibiting EGFR signaling, induces brachyury degradation repre- providing evidence of a potential clinical relevance of EGFR sents a new approach indirectly targeting this important tran- inhibitors in chordoma, but also raising questions about the scription factor with a kinase inhibitor. most suitable agent. Overall, testing EGFR inhibitors across different cell lines and We focused our studies on clinically approved EGFR inhibitors in vivo models suggests that a subset of chordomas are driven by and observed that all of them displayed some activity, although the EGFR signaling pathway and are strikingly sensitive to EGFR with different potencies, against U-CH1 and UM-Chor1 cell lines. inhibition. Preclinically, this sensitivity correlates with the level Afatinib was the most potent inhibitor in these cell lines, whereas of EGFR phosphorylation and it will be important to assess if lapatinib was the least active in addition to being inactive on the this holds true in the clinical study. Interestingly, Mug-Chor1, remaining cell lines, which might have predicted the poor activity U-CH2, and Chor-IN-1 chordoma cell lines display a strong observed for lapatinib in the clinic. Afatinib was also the only drug expression of AXL receptor, which deserves further investiga- with activity across the chordoma cell lines panel, with the tion, because it represents a mechanism of resistance in lung exception of JHC7, that despite expressing significant levels of cancer (49). Should this occur also in chordoma, a combina- EGFR is not driven by EGFR signaling. tion therapy with AXL inhibitors, which are already in the The activity of afatinib in chordoma cell lines is particularly clinic, might be beneficial to these types of patients. STK33, relevant because this drug is very selective biochemically and which is absent in the cell lines most sensitive to afatinib, also its cellular activity is dependent upon EGFR pathway activation represents a candidate biomarker for afatinib sensitivity which (41, 42). The activity of afatinib in U-CH1 and UM-Chor1 deserves further investigation. chordoma cell lines is equivalent to that reported for mutant These data provide a strong rationale for formal evaluation of EGFR-dependent lung tumors in vitro (42, 48), suggesting that afatinib in the treatment of chordoma patients. Accordingly, a chordoma might also represent a potential indication for this drug. European phase II study on afatinib in advanced chordoma is These cellular data were also corroborated by impressive in vivo about to start patient recruitment. efficacy, with tumor growth inhibition in one chordoma xenograft and in three different PDX models. Disclosure of Potential Conflicts of Interest Scheipl and colleagues (51) published a focused compound S. Stacchiotti is a consultant/advisory board (Minor) member of Bayer. No screening describing the activity of EGFR inhibitors in a subset of potential conflicts of interest were disclosed by the other authors. chordoma cell lines, reporting, in line with our data, that U-CH1 and UM-Chor1 cell lines are sensitive to EGFR inhibitors, but Authors' Contributions unexpectedly they found afatinib active on UM-Chor1 but not in Conception and design: P. Magnaghi, B. Salom, N. Amboldi, D. Ballinari, U-CH1 cell line. Considering the potent activity we repeatedly E. Tamborini, S. Pilotti, S. Stacchiotti, A. Isacchi Development of methodology: P. Magnaghi, B. Salom, L. Cozzi, N. Amboldi observed for afatinib on U-CH1 both in vitro, with modulation of Acquisition of data (provided animals, acquired and managed patients, downstream pathways, and particularly in vivo, the lack of activity provided facilities, etc.): P. Magnaghi, B. Salom, N. Amboldi, F. Gasparri, they observed in U-CH1 might be related to different screening L. Raddrizzani, C. Orrenius, F. Bozzi, J. Sommer conditions or compound handling. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Differently from erlotinib, gefitinib, and lapatinib that are computational analysis): P. Magnaghi, B. Salom, L. Cozzi, N. Amboldi, reversible inhibitors, afatinib contains an electrophilic group D. Ballinari, F. Gasparri, L. Raddrizzani, A. Somaschini, R. Bosotti, C. Orrenius Writing, review, and/or revision of the manuscript: P. Magnaghi, B. Salom, capable of Michael addition to conserved cysteines within the N. Amboldi, D. Ballinari, E. Tamborini, S. Pilotti, A. Galvani, J. Sommer, catalytic domains of EGFR (Cys797), HER2 (Cys805), and HER4 S. Stacchiotti, A. Isacchi (Cys803), that induces complete and long-lasting inhibition Administrative, technical, or material support (i.e., reporting or organizing of receptor phosphorylation (42). However, the activity of afati- data, constructing databases): P. Magnaghi, B. Salom, N. Amboldi, C. Orrenius nib in chordoma cell lines appears to be related not only to its Study supervision: P. Magnaghi, B. Salom, E. Tamborini, A. Montagnoli, higher potency and covalent mechanism, as neratinib and daco- A. Isacchi mitinib, sharing the same mechanism, were active only in U-CH1 Acknowledgments and UM-Chor1 cell lines. We acknowledge the Chordoma Foundation for providing U-CH1, U-CH2, This activity appears to be strongly contributed by its unique JHC7, and UM-Chor1 cell lines. We acknowledge the Johns Hopkins University ability to downmodulate the total level of EGFR and brachyury for JHC7 and University of Michigan for UM-Chor1 cell lines. proteins, a feature not shared by the other inhibitors. Interesting- We also acknowledge Clara Albanese, Stefano Camisasca, Antonella Land- ly, afatinib was recently shown to induce a downmodulation of onio, and Claudia Re from Nerviano Medical Sciences for technical support in fl the total level both of HER2 and EGFR in tumors harvested from ow cytometry analysis and cell culturing, and Michael Wick from START for technical support in the efficacy study. NCI-N87 xenografts treated with afatinib (52). Brachyury is a distinctive marker of sporadic chordomas, The costs of publication of this article were defrayed in part by the payment of duplication of the T gene locus is typical of familial chordomas page charges. This article must therefore be hereby marked advertisement in (6), and a DNA polymorphism in the T gene is associated with accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chordoma predisposition in the general population (53). Accord- ingly, silencing of brachyury in chordoma cell lines was widely Received April 12, 2017; revised August 31, 2017; accepted November 30, shown to impair tumor growth both in vitro and in vivo (7, 8; 2017; published OnlineFirst December 13, 2017.

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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 2018;17:603-613. Published OnlineFirst December 13, 2017.

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