The Use of Cellular Thermal Shift Assay (CETSA) to Study Crizotinib Resistance in ALK-expressing Human Cancers

Overview

Journal Sci Rep

Specialty Science

Date 2016 Sep 20

PMID 27641368

Citations 14

Authors

Abdulraheem Alshareef

Hai-Feng Zhang

Yung-Hsing Huang

Chengsheng Wu

Jing Dong Zhang

Peng Wang

Ahmed El-Sehemy

Mohamed Fares

Raymond Lai

Affiliations

Soon will be listed here.

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.

Citing Articles

Knowledge graph-based thought: a knowledge graph-enhanced LLM framework for pan-cancer question answering.

Feng Y, Zhou L, Ma C, Zheng Y, He R, Li Y Gigascience. 2025; 14.

PMID: 39775838 PMC: 11702363. DOI: 10.1093/gigascience/giae082.

Proteolysis Targeting Chimera Degraders of the METTL3-14 mA-RNA Methyltransferase.

Errani F, Invernizzi A, Herok M, Bochenkova E, Stamm F, Corbeski I JACS Au. 2024; 4(2):713-729.

PMID: 38425900 PMC: 10900215. DOI: 10.1021/jacsau.4c00040.

Targeting Trop2 by Bruceine D suppresses breast cancer metastasis by blocking Trop2/β-catenin positive feedback loop.

Tang W, Hu Y, Tu K, Gong Z, Zhu M, Yang T J Adv Res. 2023; 58:193-210.

PMID: 37271476 PMC: 10982870. DOI: 10.1016/j.jare.2023.05.012.

A superior loading control for the cellular thermal shift assay.

Delport A, Hewer R Sci Rep. 2022; 12(1):6672.

PMID: 35461337 PMC: 9035151. DOI: 10.1038/s41598-022-10653-7.

Ziprasidone suppresses pancreatic adenocarcinoma cell proliferation by targeting GOT1 to trigger glutamine metabolism reprogramming.

Yang Y, Zheng M, Han F, Shang L, Li M, Gu X J Mol Med (Berl). 2022; 100(4):599-612.

PMID: 35212782 DOI: 10.1007/s00109-022-02181-8.

References

Bresler S, Wood A, Haglund E, Courtright J, Belcastro L, Plegaria J . Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma. Sci Transl Med. 2011; 3(108):108ra114. PMC: 3319004. DOI: 10.1126/scitranslmed.3002950. View

Hegazy S, Alshareef A, Gelebart P, Anand M, Armanious H, Ingham R . Disheveled proteins promote cell growth and tumorigenicity in ALK-positive anaplastic large cell lymphoma. Cell Signal. 2012; 25(1):295-307. DOI: 10.1016/j.cellsig.2012.09.027. View

Moore N, Azarova A, Bhatnagar N, Ross K, Drake L, Frumm S . Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma. Oncotarget. 2014; 5(18):8737-49. PMC: 4226718. DOI: 10.18632/oncotarget.2372. View

Peron M, Lovisa F, Poli E, Basso G, Bonvini P . Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors. PLoS One. 2015; 10(7):e0132330. PMC: 4493009. DOI: 10.1371/journal.pone.0132330. View

Sang J, Acquaviva J, Friedland J, Smith D, Sequeira M, Zhang C . Targeted inhibition of the molecular chaperone Hsp90 overcomes ALK inhibitor resistance in non-small cell lung cancer. Cancer Discov. 2013; 3(4):430-43. PMC: 4086149. DOI: 10.1158/2159-8290.CD-12-0440. View

Bai L, Chen J, McEachern D, Liu L, Zhou H, Aguilar A . BM-1197: a novel and specific Bcl-2/Bcl-xL inhibitor inducing complete and long-lasting tumor regression in vivo. PLoS One. 2014; 9(6):e99404. PMC: 4047118. DOI: 10.1371/journal.pone.0099404. View

Sakamoto H, Tsukaguchi T, Hiroshima S, Kodama T, Kobayashi T, Fukami T . CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell. 2011; 19(5):679-90. DOI: 10.1016/j.ccr.2011.04.004. View

Amin H, McDonnell T, Medeiros L, Rassidakis G, Leventaki V, OConnor S . Characterization of 4 mantle cell lymphoma cell lines. Arch Pathol Lab Med. 2003; 127(4):424-31. DOI: 10.5858/2003-127-0424-COMCLC. View

Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H . Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007; 131(6):1190-203. DOI: 10.1016/j.cell.2007.11.025. View

10.

Togashi Y, Hayashi H, Terashima M, De Velasco M, Sakai K, Fujita Y . Inhibition of β-Catenin enhances the anticancer effect of irreversible EGFR-TKI in EGFR-mutated non-small-cell lung cancer with a T790M mutation. J Thorac Oncol. 2014; 10(1):93-101. DOI: 10.1097/JTO.0000000000000353. View

11.

Heidel F, Bullinger L, Feng Z, Wang Z, Neff T, Stein L . Genetic and pharmacologic inhibition of β-catenin targets imatinib-resistant leukemia stem cells in CML. Cell Stem Cell. 2012; 10(4):412-24. PMC: 3339412. DOI: 10.1016/j.stem.2012.02.017. View

12.

Morris S, Kirstein M, Valentine M, Dittmer K, Shapiro D, Saltman D . Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science. 1994; 263(5151):1281-4. DOI: 10.1126/science.8122112. View

13.

Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P . The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc. 2014; 9(9):2100-22. DOI: 10.1038/nprot.2014.138. View

14.

Katayama R, Friboulet L, Koike S, Lockerman E, Khan T, Gainor J . Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib. Clin Cancer Res. 2014; 20(22):5686-96. PMC: 4233168. DOI: 10.1158/1078-0432.CCR-14-1511. View

15.

Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson E, Dan C . Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science. 2013; 341(6141):84-7. DOI: 10.1126/science.1233606. View

16.

Chen Y, Takita J, Choi Y, Kato M, Ohira M, Sanada M . Oncogenic mutations of ALK kinase in neuroblastoma. Nature. 2008; 455(7215):971-4. DOI: 10.1038/nature07399. View

17.

Coluccia A, Vacca A, Dunach M, Mologni L, Redaelli S, Bustos V . Bcr-Abl stabilizes beta-catenin in chronic myeloid leukemia through its tyrosine phosphorylation. EMBO J. 2007; 26(5):1456-66. PMC: 1817619. DOI: 10.1038/sj.emboj.7601485. View

18.

Bresler S, Weiser D, Huwe P, Park J, Krytska K, Ryles H . ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma. Cancer Cell. 2014; 26(5):682-94. PMC: 4269829. DOI: 10.1016/j.ccell.2014.09.019. View

19.

Christensen J, Zou H, Arango M, Li Q, Lee J, McDonnell S . Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther. 2007; 6(12 Pt 1):3314-22. DOI: 10.1158/1535-7163.MCT-07-0365. View

20.

Griffin C, Hawkins A, Dvorak C, Henkle C, Ellingham T, Perlman E . Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res. 1999; 59(12):2776-80. View