CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

Anette Langebäck,Smaranda Bacanu,Jonas Bergh,Johan Hartman,Henriette Laursen,Wytske Van Weerden,Pär Nordlund,Takahiro Seki,Anderson Daniel Ramos,Anna Berggren,Sigrun Erkens-Schulze,Lisanne Mout,Sara Lööf,Yihai Cao

doi:10.1038/s41598-019-55526-8

Abstract

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.

Highlights

The use of taxanes has for decades been crucial for treatment of several cancers
Melt curves for α- and β-tubulin were generated in K562-cells, a suspension cell line originating from myelogenous leukemia
We recently described the correlation of Cellular Thermal Shift Assays (CETSAs) melt curves of interacting proteins and named this phenomena thermal proximity co-aggregation (TPCA)[25,26], which is the likely explanation for the correlated shifts www.nature.com/scientificreports

Summary

Introduction

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. 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. The addition of docetaxel in parallel to androgen deprivation therapy was recently shown to increase overall survival, suggesting that an earlier switch to taxane regime might be favourable in these cases[8] The taxanes exert their effect by binding to β-tubulin on the luminal side of microtubules leading to the stabilization of microtubules via inhibition of depolymerization[9]. Other general mechanisms for resistance to cytotoxic drugs have been suggested to affect taxane sensitivity such as altered expression of anti-apoptotic proteins (e.g. Bcl-2 and Bcl-xL)[15,16] and NFkB modulation[17]

Methods

Results

Conclusion