The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.

Abdulraheem Alshareef,Raymond Lai,Ahmed El-Sehemy,Peng Wang,Hai-Feng Zhang,Yung-Hsing Huang,Jing Dong Zhang,Mohamed Fares,Chengsheng Wu

doi:10.1038/srep33710

Abstract

Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners.

Highlights

cellular thermal shift assay (CETSA) at 52 °C Positive binding Positive binding Positive binding Negative binding Negative binding Negative binding Negative binding therapeutically efficacious in treating Anaplastic lymphoma kinase (ALK)+ non-small cell lung cancers (NSCLC) patients, many of the treated patients showed disease progression within a year of therapy[16]
In NSCLC, while it was initially reported that the differential Crizotinib sensitivity in echinoderm microtubule-associated protein like 4 (EML4)-ALK-expressing cells is related to the existence of the four EML4-ALK fusion variants[19], results from subsequent studies did not confirm the relationship between these EML4-ALK variants and Crizotinib responses[17,20]
Our results have led us to conclude that the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity in ALK+ cancer cells, and Crizotinib—ALK binding is in turn dictated by structure of ALK and some of its binding partners

Summary

Introduction

CETSA at 52 °C Positive binding Positive binding Positive binding Negative binding Negative binding Negative binding Negative binding therapeutically efficacious in treating ALK+ NSCLC patients, many of the treated patients showed disease progression within a year of therapy[16]. The mechanisms underlying the differential clinical responses to Crizotinib are not well understood. In NSCLC, while it was initially reported that the differential Crizotinib sensitivity in EML4-ALK-expressing cells is related to the existence of the four EML4-ALK fusion variants[19], results from subsequent studies did not confirm the relationship between these EML4-ALK variants and Crizotinib responses[17,20]. Resistance to Crizotinib remains to a significant challenge in the clinic, and the mechanisms underlying this specific drug resistance is incompletely understood. Our results have led us to conclude that the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity in ALK+ cancer cells, and Crizotinib—ALK binding is in turn dictated by structure of ALK and some of its binding partners

Objectives

Methods

Results

Conclusion