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The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development. We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug action can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues. Although CETSA was first developed for evaluating drug binding to target proteins in cells it is also useful for characterizing physiological interactions, including protein-protein, protein-metabolite and protein-nucleic acid interactions (Martinez Molina & Nordlund, Ann Rev Pharm Toxic 2016 56:141). In the proteome-wide CETSA experiment, interactions with more than 7000 proteins can be measured in parallel (Savitski, Science 2014 346;6205). This strategy therefore constitutes a novel mean for discovering key interactions determining the fate of cancer cells during therapy and resistance – interactions that can serve as biomarkers or candidates for novel drug targets in cancer therapy. Citation Format: Ying Yu Liang, Anderson Ramos, Henriette Laursen, Olga Surova, Johan Lengqvist, Sara Lööf, Anette Langebäck, Smaranda Bacanu, Jonas Bergh, Pär Nordlund. Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5655.