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. 2016 Jul;239(3):320-34.
doi: 10.1002/path.4729. Epub 2016 May 31.

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen

Susanne Scheipl  1   2 , Michelle Barnard  1   3   4 , Lucia Cottone  1 , Mette Jorgensen  1 , David H Drewry  5   6 , William J Zuercher  5   6 , Fabrice Turlais  3 , Hongtao Ye  7 , Ana P Leite  1 , James A Smith  3 , Andreas Leithner  2 , Peter Möller  8 , Silke Brüderlein  8 , Naomi Guppy  9 , Fernanda Amary  7 , Roberto Tirabosco  7 , Sandra J Strauss  1 , Nischalan Pillay  1   7 , Adrienne M Flanagan  1   7   9
Affiliations

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen

Susanne Scheipl et al. J Pathol. 2016 Jul.

Abstract

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50 ) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: AZD8931; EGFR; ERBB family; chordoma; drug screen; resistance.

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Figures

Figure 1
Figure 1
An overview of the screening cascade
Figure 2
Figure 2
Hit compounds display varying effects on p‐EGFR and EGFR levels. Of 21 EGFR/ERBB hit compounds that selectively targeted chordoma cells, the impact of 13, comprising a selection of hit compounds across the libraries and chemical structures tested (listed in Tables 1, 2), was studied by western blot on three chordoma cell lines (U‐CH1, U‐CH2, MUG‐Chor1). Cells were serum‐starved overnight before being treated with EGFR inhibitors (250 nm) for 4 h and then being exposed to EGF (50 ng/ml) for 15 min.
Figure 3
Figure 3
Western blot (A) and ELISA (B) analysis confirm suppression of the biomarker p‐EGFR upon treatment with EGFR TKIs in U‐CH1 and UM‐Chor1. Cells were serum‐starved overnight before they were treated with a range of concentrations of the EGFR inhibitors for 4 h and then EGF‐spiked (50 ng/ml) for 15 min. Endogenous controls (non‐serum‐starved, non‐EGF‐spiked), untreated controls (serum‐starved, non‐EGF‐spiked) and vehicle controls (serum‐starved, treated with 2.5% DMSO, EGF‐spiked) were included. Phospho‐EGFR was measured by western blot and ELISA. Western blot results for U‐CH2, U‐CH7, JCH7 and MUG‐Chor1 are displayed in supplementary material, Figure S2. (C, D) Sapitinib induces a significant growth reduction in the patient‐derived xenograft SF8894 (C) and in the U‐CH1 xenograft (D); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001
Figure 4
Figure 4
Apoptotic induction in U‐CH1 (A) and UM‐Chor1 (B). The Caspase‐Glo® 3/7 Assay and the CellTiter‐Glo® Luminescent Cell Viability Assay were used on separate assay plates to monitor cell viability and to determine induction of apoptosis upon treatment with erlotinib, gefitinib, sapitinib, afatinib and lapatinib. Read‐outs were performed at four time points (6, 24, 48 and 72 h). Two independent experiments were conducted for each compound (n = 3 for sapitinib and erlotinib). The results for U‐CH7 and MUG‐Chor1 are shown in supplementary material, Figure S6
Figure 5
Figure 5
MET expression in the chordoma cell line panel. (A) Western blot analyses for MET‐expression in the cell line panel were conducted on endogenous (non‐serum‐starved, non‐EGF‐spiked), serum‐starved (serum‐starved, non‐EGF‐spiked) and EGF‐spiked (serum‐starved, EGF‐spiked) samples of each chordoma cell line. Normal adult human dermal fibroblasts (NAHDF) served as a control. Both western blots and immunohistochemistry (data shown in supplementary material, Figure 6B) revealed strong p‐MET expression in U‐CH2, a cell line resistant to EGFR TKIs, but not in the other chordoma cell lines. (B) Western blots of U‐CH2 treated with reagents as indicated for 4 h
Figure 6
Figure 6
Combination treatment of the EGFR TKI sapitinib and the MET inhibitor crizotinib revealed significant synergy. (A) U‐CH2 cells were plated with a Multidrop Combi in a 384‐well format: after 24 h, cells were treated with crizotinib for 72 h, followed by sapitinib for another 24 h (n = 4 for combination; n = 3 for compounds alone); a combination index (CI) was calculated and evaluated as synergistic (CI < 0.9), additive (CI = 0.9–1.1) or antagonistic (CI > 1.1) 86; we observed a significant synergistic effect when sapitinib (300 nm) was combined with the MET inhibitor crizotinib (1 µm) in U‐CH2 (MI 58%; CI = 0.121; combination versus control, **p = 0.0047). (B) Immunohistochemistry was conducted on formalin‐fixed, paraffin‐embedded pellets of all seven chordoma cell lines, normal adult human dermal fibroblasts (NAHDF) and positive controls (POS): all images were taken at × 20 magnification; results for p‐MET showed strong expression in U‐CH2, concordant with the results obtained in western blot analysis (Figure 5), but not in the other cell lines. (C) PTEN expression was absent in U‐CH1, weak in UM‐Chor1 and positive to varying degrees in the other cell lines. (D) E‐Cadherin was expressed weakly and only focally in U‐CH7 and MUG‐Chor1 and was negative in the remaining five cell lines. (E) Western blots on the chordoma cell line panel (n = 7) and NAHDF confirmed an absence of PTEN in U‐CH1, weak expression in UM‐Chor1 and varying positivity in the other cell lines, as observed in IHC (C)
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