Abstract B29: Combinatorial screens with targeted inhibitors reveal diverse compensatory responses and mechanisms of adaptive resistance to therapy
Authors: Axelrod Mark J. , Roller Devin , Capaldo Brian , Mackey Aaron J. , Conaway Mark , Gioeli Daniel , Weber Michael J. 
Published: August 2014

The effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling, which generates adaptive resistance mechanisms and reduces therapeutic responses. We have performed high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that confer resistance to the cytotoxic effects of targeted therapy. We screened subsets of over 1,000 drug combinations in 14 different epithelial cell lines representing three distinct cancer lineages, and 19 melanoma cell lines, and assessed the ability of each combination to cause synergistic cytotoxicity. We focused on synergistic combinations because they point to mechanistic linkages between the signaling pathways, and also because of the possibility of improved therapeutic index in vivo. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple epithelial cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern with respect to lineage specificity or mutational status of commonly altered oncogenes in the observed synergies. Within the melanoma panel, BRAF mutational status predicted response to single-agent BRAF inhibition, but did not predict synergistic drug combinations, which were different for each cell line. These results reflect the heterogeneity of genetic alterations and the plasticity of cell signaling networks even among cell lines of the same tissue of origin that contain the same predominant driver mutations. We suggest that there is not a sharp dichotomy between “driver” and “passenger” mutations, and that the biological responses to combination therapies are determined by functionally important modifier mutations that we term “back-seat drivers.” This hypothesis is supported by analysis of the transcriptomes and phosphoproteomes of cells treated with drugs singly and in combination, and by exome sequencing. Such analyses also can reveal critical nodes with the potential to function as effective single targets. We found that co-inhibition of EGFR and PI3 Kinase causes synergistic cytotoxicity in some epithelial cancer cell lines, and that phosphoproteomic analysis of signaling pathway responses revealed concordant synergistic inhibition of p70S6 Kinase in KU-7 bladder cancer cells. Using an epistasis paradigm, restoration of p70S6 Kinase signaling by expression of mutationally activated p70S6 Kinase resulted in protection from cytotoxicity, indicating that p70S6 Kinase is a critical node for enhanced cytotoxicity due to combination treatment. AT7867, a potent inhibitor of p70S6 Kinase, was able to inhibit phosphorylation of ribosomal protein S6 and induce cytotoxicity as effectively as the combination drug treatment. We suggest that p70S6 Kinase acts as a functionally important node within the EGFR/PI3 Kinase signaling network and is an attractive target for therapeutic intervention.

Citation Format: Mark J. Axelrod, Devin Roller, Brian Capaldo, Aaron J. Mackey, Mark Conaway, Daniel Gioeli, Michael J. Weber. Combinatorial screens with targeted inhibitors reveal diverse compensatory responses and mechanisms of adaptive resistance to therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B29.