title: The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.
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.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.|Department of Applied Medical Sciences, Taibah University, Almedinah, P.O. Box 41477, Saudi Arabia.
authors.forename: Abdulraheem
authors.initials: A
authors.lastname: Alshareef
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.|Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V5Z 1L3, Canada.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Zhang
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yung-Hsing
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Chengsheng
authors.initials: C
authors.lastname: Wu
authors.affiliation: Department of Medical Oncology, The First hospital of China Medical University, Shen Yang 110001, P. R. China.
authors.forename: Jing Dong
authors.initials: JD
authors.lastname: Zhang
authors.affiliation: Department of Internal Medicine, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
authors.forename: Ahmed
authors.initials: A
authors.lastname: El-Sehemy
authors.affiliation: National Research Center, Cairo, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Fares
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.|Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.|DynaLIFEDx Medical Laboratories, Edmonton, Canada.
authors.forename: Raymond
authors.initials: R
authors.lastname: Lai
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D000077547:Crizotinib; D019008:Drug Resistance, Neoplasm; D015870:Gene Expression; D006801:Humans; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D034741:RNA, Small Interfering; D051176:beta Catenin; D000077547:Crizotinib; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases