Application of Kinase Stable Cell Lines Detection of Secondary EGFR Mutations in Afatinib-Resistant BaF3 Cells

Kinases serve as key regulatory molecules for most cellular pathways, which are often associated with diseases: as causative agents for diseases, especially many cancers, or as targets for drug development. These targets include epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2), non-receptor tyrosine kinase (ABL), etc.

Gene function analysis, target discovery and validation, assay development and compound screening often require cell-based assays. Stable cell lines that express target genes through transgene integration into the host genome provide an efficient way to perform such analyses. At Creative Biogene, we offer a wide range of kinase stable cell lines that can help researchers save time and cost by shifting the tedious process of cell line generation and validation to the study of major questions such as target discovery and drug development.

Construction of EGFR Mutant Cell Lines

Ba/F3 is a murine interleukin-3 dependent pro-B cell line known as a model system for assessing downstream signaling of kinase oncogenes and the ability of small molecule kinase inhibitors to block kinase activity. Creative Biogene has developed a series of EGFR mutant BaF3 stable cell lines by introducing EGFR mutations into Ba/F3 cells through retroviral transduction. The cell lines were mainly selected with GFP and subsequently validated by PCR sequencing and finally confirmed by protein blotting. Clones with the highest expression levels have been introduced to facilitate drug screening and related studies.

Available EGFR Mutant BaF3 Stable Cell Lines
Human EGFR Stable Cell Line-Ba/F3	Human EGFR-C797S Stable Cell Line-Ba/F3	Human EGFR-D770_N771insSVD Stable Cell Line-Ba/F3
Human EGFR-DEL19 Stable Cell Line-Ba/F3	Human EGFR-DEL19/C797S Stable Cell Line-Ba/F3	Human EGFR-DEL19/T790M Stable Cell Line-Ba/F3
Human EGFR-DEL19/T790M/C797S Stable Cell Line-Ba/F3	Human EGFR-G719S Stable Cell Line-Ba/F3	Human EGFR-G719S/T263P Stable Cell Line-Ba/F3
Human EGFR-H773_V774insNPH Stable Cell Line-Ba/F3	Human EGFR-L858R Stable Cell Line-Ba/F3	Human EGFR-L858R/C797S Stable Cell Line-Ba/F3
Human EGFR-L858R/T790M Stable Cell Line-Ba/F3	Human EGFR-L858R/T790M/C797S Stable Cell Line-Ba/F3	Human EGFR-L861Q Stable Cell Line-Ba/F3
Human EGFR-T790M Stable Cell Line-Ba/F3	Human EGFR-T790M/C797S Stable Cell Line-Ba/F3	Human EGFR-T790M/L861Q Stable Cell Line-Ba/F3
Human EGFR-Del19/T790M/L858R Stable Cell Line-Ba/F3	Human EGFR-P699_N700insASV Stable Cell Line - BaF3	Human EGFR-V769_D770insASV Stable Cell Line - BaF3
Human EGFR-VIII Stable Cell Line - BaF3	Human EGFR_S768I Stable Cell Line - BaF3	Human EGFR-A289T Stable Cell Line - BaF3
Human EGFR-A289D Stable Cell Line - BaF3	Human EGFR-A289V Stable Cell Line - BaF3	Human EGFR-A763_Y764insFQEA Stable Cell Line - BaF3
Human EGFR-D770_N771insG Stable Cell Line - BaF3	Human EGFR-N771-P772_ins_H Stable Cell Line - BaF3	Human EGFR-N771del_ins_GY Stable Cell Line - BaF3
Human EGFR-D770del_ins_GY Stable Cell Line - BaF3	Human EGFR-H773-V774_ins_H Stable Cell Line - BaF3	Human EGFR-V774-C775_ins_HV Stable Cell Line - BaF3
Human EGFR-H773-V774_ins_AH Stable Cell Line - BaF3	Human EGFR-D770-N771_ins_Y Stable Cell Line - BaF3	Human EGFR-D770-N771_ins_GT Stable Cell Line - BaF3
Human EGFR-N771-P772_ins_N Stable Cell Line - BaF3	Human EGFR-I744-K745_ins_KIPVAI Stable Cell Line - BaF3	Human EGFR-P772-H773_ins_NP Stable Cell Line - BaF3
Human EGFR-K745-E746_ins_VPVAIK Stable Cell Line - BaF3	Human EGFR-V769-D770_ins_GE Stable Cell Line - BaF3	Human EGFR-K745-E746_ins_IPVAIK Stable Cell Line - BaF3
Human EGFR-V769-D770_ins_GE Stable Cell Line - BaF3	Human EGFR-K745-E746_ins_VPVAIK Stable Cell Line - BaF3	Human EGFR-D770-N771_ins_GF Stable Cell Line - BaF3
Human EGFR-A767-S768_ins_TLA Stable Cell Line - BaF3	Human EGFR-D770-N771_ins_NPG Stable Cell Line - BaF3	Human EGFR-H773-V774_ins_PH Stable Cell Line - BaF3
Human EGFR-Y764-V765_ins_HH Stable Cell Line - BaF3	Human EGFR_G724S Stable Cell Line - BaF3	Human EGFR-P772-H773_ins_NPH Stable Cell Line - BaF3

Research Applications

EGFR mutant BaF3 stable cell lines have a wide range of applications in the following areas, notably:

Investigating the effects of EGFR mutations on downstream signaling pathways and cellular processes, such as cell proliferation, differentiation, and survival.
Identifying novel therapeutic strategies for targeting EGFR mutants in cancer by testing the efficacy of various inhibitors or combination therapies.
Screening for small molecule compounds or antibodies that selectively target EGFR mutants over wild-type EGFR.
Developing in vitro and in vivo models to study the mechanisms of resistance to EGFR inhibitors and to test novel combination therapies.

Mechanisms of Acquired Resistance to Afatinib in Lung Cancer

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI). Tumors with exon 18 mutations are particularly sensitive to irreversible second-generation (2G) afatinib compared with first-generation TKIs (1G-TKI). However, data on the mechanisms of acquired resistance to afatinib are limited.

Afatinib-resistant cells were established by transfecting Ba/F3 cells with common or exon 18 (G719A and Del18) mutations and exposing them to increasing concentrations of afatinib. Afatinib-resistant clones were separately established through N-ethyl-N-nitrosourea (ENU) mutagenesis and exposure to fixed concentrations of afatinib. Rebiopsy samples from patients whose tumors acquired resistance to afatinib were analyzed.

Fig. 1 Secondary EGFR mutations of afatinib-resistant Ba/F3 cells and NSCLC cell lines. (Kobayashi, et al., 2017)

Afatinib-resistant cells with Del19, L858R, or G719A developed T790M, whereas those with Del18 acquired novel L792F mutation. ENU mutagenesis screening established 84 afatinib-resistant clones. All Del19 clones and most of the other clones acquired only T790M. However, C797S occurred in subsets of L858R, G719A, and Del18 clones. In addition, subsets of Del18 clones acquired L792F. C797S-acquired cells were sensitive to 1G erlotinib. L792F demonstrated intermediate resistance between T790M and C797S to both 1G- and 3G-TKIs, whereas L792F was the least resistant to 2G-TKIs, particularly dacomitinib. Chronic exposure of Del18 + L792F cells to dacomitinib induced additional T790M. T790M was detected in one of four clinical samples. In conclusion, L792F and C797S, in addition to the major T790M, can develop in afatinib-resistant cells particularly using a low dose of afatinib, and these minor mutations appear to exhibit sensitivity to dacomitinib and erlotinib, respectively. These data suggest that detection of these secondary mutations is important to overcome acquired resistance to afatinib.

Reference:

Kobayashi, Y.; et al. Characterization of EGFR T790M, L792F, and C797S Mutations as Mechanisms of Acquired Resistance to Afatinib in Lung Cancer. Mol Cancer Ther. 2017 Feb;16(2):357-364.

* For research use only. Not intended for any clinical use.

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Application of Kinase Stable Cell Lines Detection of Secondary EGFR Mutations in Afatinib-Resistant BaF3 Cells

Construction of EGFR Mutant Cell Lines

Research Applications

Mechanisms of Acquired Resistance to Afatinib in Lung Cancer

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