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Supplemental Figure Legends
Figure S1. Fifty percent (50%) inhibitory concentration of neratinib, afatinib and dacomitinib in Ba/F3
cells expressing different EGFR or HER2 exon 20 insertion mutations and the EGFR exon 20 insertion
patient derived cell lines. to second generation EGFR-TKIs.
Figure S2. Efficacy of neratinib, afatinib or dacomitinib against HER2 exon 20 insertion mutants
measured in a soft agar assay. A. NIH-3T3 cells expressing the respective mutations were suspended in
soft agar with increasing concentrations of each drug for a colony formation assay and photographed after
2 weeks of incubation. Photomicrographs show the result of 0.01 M of each drug. Left sided columns
are at low magnification and right columns are at high magnification. B. The mean and SD of colony
numbers are plotted for each drug concentration for the different HER2 exon 20 insertion mutants.
Figure S3. Patient derived EGFR exon 20 insertion cell lines. A. Morphological features of patientderived cell lines DFCI58 and DFCI127. B. DFCI58 and DFCI127 were treated with different drugs at
the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted
relative to untreated controls. C. DFCI58 and DFCI127 were treated with gefitinib or dacomitinib at
indicated concentrations for 6 hours or 48 hours (bottom panel only). Cell extracts were immunoblotted to
detect the indicated proteins.
Figure S4. Comparison of amino acid sequences of exon 20 of EGFR to HER2. This alignment was
obtained using BLAST (basic local alignment search tool), and both amino acid sequences were obtained
from GenBank (accession number: EGFR at NM_005228.3, HER2 at NM_004448.2). The sequence of
EGFR is on upper line and HER2 is on lower line. The numbers at each end are codon numbers. Codon
797 of EGFR and codon 805 of HER2 are indicated in red.