Abstract 3579: Identification of biomarkers of AXL-mediated drug resistance in head and neck squamous cell carcinoma
Authors: Balaji Kavitha , Cardnell Robert , Diao Lixia , Tong Pan , Mak Milena , Fan You Hong , Masrorpour Fatemeh , Warner Steven L. , Bearss David J. , Wistuba Ignacio , Mills Gordon B. , Heymach John , Keyomarsi Khandan , Wang Jing , Byers Lauren Averett 
Published: August 2015

Abstract
Background:
Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer. In 2013, there were ∼53,000 newly diagnosed cases and ∼11,000 deaths related to HNSCC in the USA. Overexpression of EGFR is seen in 90% HNSCC; but, only ∼10% of patients treated with the anti-EGFR antibody cetuximab show increased response rates to cetuximab and these eventually gain resistance by poorly-characterized mechanisms. We showed an association between EMT and resistance to EGFR inhibitors in lung cancers (LC) and HNSCC using a 76-gene EMT signature. AXL was identified as a therapeutic candidate linking EMT and drug resistance, showing significantly higher expression in erlotinib resistant cell lines. Other groups have linked AXL to drug resistance in HNSCC, LC and breast cancers. Here we identify signaling pathways that are regulated by AXL, mediate drug resistance, and identify potential therapeutic targets to combine with AXL inhibition.
Methods:
Using 6 clinical cohorts including The Cancer Genome Atlas (TCGA N = 493) and PROSPECT (N = 142) across 3 cancer types, we identified genes whose mRNA expression was highly correlated with AXL. Protein profiling by reverse phase protein array (RPPA) to analyze total and phospho-proteins in HNSCC cell lines, pre- and post-AXL inhibitor treatment was used to identify pathways altered upon AXL inhibition. The response to AXL inhibition was assayed in HNSCC cell lines by proliferation assays and correlated to mRNA and protein expression.
Results:
Using gene-expression and RPPA analysis we saw the highest association of AXL with pathways involved in EMT (TGF-β, Rho GTPases), autophagy and immune response. Following treatment with an AXL inhibitor, we observed a decrease in phospho-proteins in the PI3K-AKT pathway, increased expression of markers associated with apoptosis, an epithelial phenotype, and p-EGFR. Using an AXL knockdown model system in HNSCC cell lines, we validated an increase in EGFR signaling (EGFR and p-Erk), epithelial (E-cadherin), apoptotic (cleaved PARP and caspase-7) and DNA repair proteins (RAD51, ku-80 and PARP) and a decrease in Slug, Twist and ZEB-1, indicating that AXL may be directly involved in mediating EMT. AXL knockdown reduced proliferation of HNSCC cell lines and AXL inhibition was able to re-sensitize resistant HNSCC cell lines to erlotinib, an EGFR tyrosine kinase inhibitor.
Conclusions:
In summary, we identified potential therapeutic targets that are upregulated with AXL expression in HNSCC and LC patient tumors and cell lines. Using AXL inhibitor and knockdown in HNSCC cell lines, we validated biomarkers involved in EMT, EGFR signaling and apoptosis that are altered upon AXL inhibition. AXL inhibition led to an epithelial phenotype in cells and re-sensitized resistant cells to erlotinib. Studies are ongoing to validate the mechanisms of AXL-mediated drug resistance and to identify potential combination treatments that can synergize with AXL-inhibition.
Citation Format: Kavitha Balaji, Robert Cardnell, Lixia Diao, Pan Tong, Milena Mak, You Hong Fan, Fatemeh Masrorpour, Steven L. Warner, David J. Bearss, Ignacio Wistuba, Gordon B. Mills, John Heymach, Khandan Keyomarsi, Jing Wang, Lauren Averett Byers. Identification of biomarkers of AXL-mediated drug resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3579. doi:10.1158/1538-7445.AM2015-3579