Small Molecule Therapeutics Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury Paola Magnaghi 1 , Barbara Salom 1 , Liviana Cozzi 1 , Nadia Amboldi 1 , Dario Ballinari 1 , Elena Tamborini 2 , Fabio Gasparri 1 , Alessia Montagnoli 1 , Laura Raddrizzani 1 , Alessio Somaschini 1 , Roberta Bosotti 1 , Christian Orrenius 1 , Fabio Bozzi 2 , Silvana Pilotti 2 , Arturo Galvani 1 , Josh Sommer 3 , Silvia Stacchiotti 2 , and Antonella Isacchi 1 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 col- lected clinically approved drugs and other advanced inhibitors of MET, PDGFRb, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRb inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insen- sitive 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 antiproliferative IC 50 s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chor- doma. 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. Mol Cancer Ther; 17(3); 603–13. Ó2017 AACR. 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% to 4% of all primary bone malignancies. They are typically late onset tumors with a peak incidence between the fifth and sixth decades 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 metastases occur in 20% to 30% of cases (1), but local recurrences affect >50% of patients (2). In case of relapse, surgery or radiotherapy 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 devel- opment (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 (7–9). Multiple studies have shown that chordomas commonly exhibit expres- sion and activation of tyrosine kinase receptors and downstream signaling molecules, with MET (HGF receptor), PDGFRb (PDGFRB), and EGFR as the most widely expressed, and HER2 (ERBB2), KIT (SCFR), and VEGFR (KDR) also expressed (10–14). 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– 19). Imatinib demonstrated some clinical benefit, although not achieving dimensional and long-lasting responses (16), 1 Oncology, Nerviano Medical Sciences, Nerviano, Milan, Italy. 2 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. 3 Chordoma Foundation, Durham, North Carolina. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Paola Magnaghi, Nerviano Medical Sciences, Viale L. Pasteur 10, Nerviano, Milan, Italy. Phone: þ390331581287; Fax: þ390331581267; E-mail: paola.magnaghi@nervianoms.com doi: 10.1158/1535-7163.MCT-17-0324 Ó2017 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 603 on May 23, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0324