Table S1 contains IC50 values for cetuximab inhibition, EGFR mutation and amplification status of tested cell lines. Figure S1 shows cetuximab binding to surface EGFR on HN5 and cetuximab resistant HN5 cell lines. Figure S2 shows EGFR levels in parental HN5 cells and cetuximab resistant clones. Figure S3 Overexposed image of immunoblot presented in Figure 2B. Figure S4 shows EGFR degradation upon treatment with anti-EGFR 2mixture for 48h. Figure S5 shows LNA-mediated knockdown of EGFR inhibit growth and proliferation of HN5, HN5 CR2, and HN5 CR14. Figure S6 EGFR levels in FaDu versus cetuximab resistant FaDu cell lines. Figure S7 HER3 and IGF1R plasticity as a mechanism of acquired cetuximab resistance in HN5 CR14. Figure S8 Full dose-response curves of cetuximab and EGFR+HER3+IGF1R 5 mixture in a panel of SCCUAT cell lines.
ARTICLE ABSTRACTSquamous cell carcinomas (SCC) arising in upper parts of the aerodigestive tract are among the leading causes of death worldwide. EGFR has been found to play an essential role in driving the malignancy of SCC of the upper aerodigestive tract (SCCUAT), but, despite this, clinical results using a range of different EGFR-targeted agents have been disappointing. Cetuximab is currently the only EGFR-targeted agent approved by the FDA for treatment of SCCUAT. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to better understand the underlying mechanisms of cetuximab resistance in SCCUAT, we established from cetuximab-sensitive models cell lines with acquired resistance to cetuximab by continuous selective pressure in vitro and in vivo. Our results show that resistant clones maintain partial dependency on EGFR and that receptor tyrosine kinase plasticity mediated by HER3 and IGF1R plays an essential role. A multitarget mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance in vitro. To our surprise, these findings could be extended to include SCCUAT cell lines with intrinsic resistance to cetuximab, suggesting that the triad consisting of EGFR, HER3, and IGF1R plays a key role in SCCUAT. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in SCCUAT. Mol Cancer Ther; 15(7); 1614–26. ©2016 AACR.