Ba/F3 isogenic preclinical model of EGFR exon 20 insertions mutations to probe EGFR TKIs, including mobocertinib. (A) The therapeutic window of different EGFR TKIs to a set of EGFR exon 20 mutations. Cells were plated at a density of 5000 cells per well (96-well plates) and grown over 3 days after treatment. The logarithm of the IC50 of EGFR exon 20 mutants compared with EGFR-WT is plotted (three separate experiments are used to generate IC50). Values below zero indicate sensitivity, whereas values above zero indicate resistance to EGFR TKIs. (B) Dose-response proliferation assays (proportional percent viability) of osimertinib for all tested EGFR mutants. (C) Dose-response proliferation assays (proportional percent viability) of poziotinib for all tested EGFR mutants. (D) Dose-response proliferation assays (proportional percent viability) of mobocertinib for all tested EGFR mutants. (E) Western blotting of Ba/F3 cells driven by EGFR-L858R and EGFR-A767_V769dupASV. Cells were treated with the EGFR TKI mobocertinib for 8 hours at the indicated ascending concentrations. pEGFR at position 1068, total EGFR, and β-actin as a loading control are displayed. IC50, 50% inhibitory concentration; pEGFR, phosphorylated EGFR; TKI, tyrosine kinase inhibitor; WT, wildtype.