Mutagenesis Screen for EGFR Inhibitor Resistance

N-ethyl-N-nitrosourea (ENU) was purchased from Sigma Aldrich and mutagenesis studies were carried as previously described (25 (link)). Briefly, 1×10⁶ cells/ml of L858R and L858R/T790M Ba/F3 cells were treated with 50μg/ml of ENU for 24 hours before the cells were washed in RPMI media and allowed to expand. 1×10⁴ cells per well were plated in 96 wells and 5 plates were plated per condition. These cells were treated continuously with either DMSO, 1μM gefitinib, 1μM osimertinib, 10μM JBJ-04-125-02 alone or with gefitinib/JBJ or osimertinib/JBJ drug combinations with media and drug change once a week. Cell growth was monitored and number of resistant colonies were counted and expanded.

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To C., Jang J., Chen T., Park E., Mushajiang M., De Clercq D.J., Xu M., Wang S., Cameron M.D., Heppner D.E., Shin B.H., Gero T.W., Yang A., Dahlberg S.E., Wong K.K., Eck M.J., Gray N.S, & Jänne P.A. (2019). Single and dual targeting of mutant EGFR with an allosteric inhibitor. Cancer discovery, 9(7), 926-943.

Publication 2019

N-ethyl-N-nitrosourea (ENU)

Cell Dmso Drug Drug combinations Gefitinib Mutagenesis N ethyl n nitrosourea Nitrosourea Osimertinib

Corresponding Organization :

Other organizations : Dana-Farber Cancer Institute, Harvard University, Scripps Research Institute

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Variable analysis

independent variables

Treatment with 50μg/ml of ENU for 24 hours
Treatment with DMSO
Treatment with 1μM gefitinib
Treatment with 1μM osimertinib
Treatment with 10μM JBJ-04-125-02
Treatment with gefitinib + JBJ
Treatment with osimertinib + JBJ

dependent variables

Number of resistant colonies
Cell growth

control variables

L858R and L858R/T790M Ba/F3 cell lines
Plating 1×10^4 cells per well in 96 wells
Plating 5 plates per condition
Media and drug change once a week

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