Abstract A122: Pre-clinical investigation of resistance to molecular targeted therapeutics in head and neck cancer

Abstract

Head and neck squamous cell carcinoma remains a deadly disease with few successful treatments available in the recurrent and/or metastatic setting. The EGFR targeting monoclonal antibody cetuximab improves survival for some patients, but either intrinsic or acquired resistance develops in nearly all cases. Small molecule inhibitors targeting the PI3K/Akt/mTORC signaling axis downstream of EGFR and other RTKs have been proposed for use in combination with cetuximab, but have thus far produced underwhelming results in the clinical setting. In this work, using a variety in vitro and in vivo of pre-clinical models, we have evaluated this potential therapeutic combination, and attempted to identify additional mechanisms of resistance. An array of head and neck squamous cell carcinoma (HNSCC) cell lines, including both human papillomavirus (HPV) positive and negative lines were utilized. The responses to both PI3K and mTORC inhibitors, alone and in combination with cetuximab was evaluated using proliferation and colony formation assays. Target inhibition and downstream signaling pathways were investigated using western blot and immunohistochemistry. Patient derived xenografts (PDXs) were used to evaluate treatment combinations in vivo following targeted sequencing to define mutational profiles in common cancer associated genes. Cetuximab decreased activation of the MAPK signaling pathway, but caused less dramatic supression of Akt signaling, and produced only modest growth inhibition. Treatment with the mTORC inhibitor AZD8055 or the dual PI3K/mTORC inhibitor NVP-BEZ-235 effectively inactivated both Akt signaling and produced much greater growth inhibition without effecting MAPK pathways. In vitro, combination therapy with cetuximab and either AZD8055 or NVP-BEZ-235 produced little additional benefit. We sought to test these compounds in our PDX model by first identifying tumors likely to be responsive to them. Sensitivity to cetuximab was determined by treating tumors with 4 doses of cetuximab or control over 2 weeks. To identify potential proteomic biomarkers that would predict response to cetuximab, IHC staining of pre-treatment tumor samples was carried out and demonstrated that high phospho-Akt and phospho-ERK levels correlated with cetuximab resistance. Several tumors that displayed poor response to cetuximab were treated with the mTORC inhibitor AZD8055 either alone, or in combination with cetuximab. Combination therapy produced marked improvement in growth inhibition compared with either drug alone. Our results are consistent with previous in vitro studies that have demonstrated the effectiveness of co-targeting EGFR and the PI3K/Akt/mTORC axis and suggest that combination therapy can be effective in appropriately selected patients. Beyond the use of pre-treatment biomarkers, we are currently investigating additional cellular processes that may provide mechanisms of resistance to EGFR and/or mTORC inhibition. These ongoing studies may provide additional therapeutic targets for future studies. Citation Format: Adam Swick, Jyothiprashanth Prabakaran, Michael Fisher, Andrew Stein, Dana Gunderson, Kwangok Nickel, Randall Kimple. Pre-clinical investigation of resistance to molecular targeted therapeutics in head and neck cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A122.