Cell lysis, SDS PAGE and Western blotting were done as described previously. The following antibodies were used: phosphorylated c-Met inhibitor in clinical trials ERBB2 Tyr1248, ERBB2 Tyr1221/1222, ERBB2, p44/42 mitogen activated protein kinase , phosphospecific ERK1/ERK2, pStat5 Tyr694, Stat5, p SAPK/JNK , SAPK/JNK, pAKT , and AKT1/2. Bands were visualized using the enhanced chemiluminescence system. Anchorage independent cell growth was analysed by colony formation ability in soft agar assay as described previously. Analysis of cell proliferation was done using an 3 5 based method by absorption of formazan at 490 nm. Samples were measured in triplicates after 48 h of culture in indicated drug concentrations. Lapatinib resistance screen Ba/F3 cells stably expressing wild type ErbB2 were treated twice with 100 mg/mL of N ethyl N nitrosourea for 12 hours.
Cells were then washed thoroughly and cultured in 96 well plates at a density of 46105 per well in the presence of 2 mM lapatinib. Lapatinib resistant cell colonies were isolated. Total RNA was extracted using TRIzol reagent. cDNA encompassing BMS-540215 ErbB2 kinase domain was synthesized by one step reverse transcription PCR and sequenced. Structural analysis of lapatinib resistant ERBB2 mutants Crystal structure coordinates for inhibitor complexes with the ErbB1 kinase domain, ErbB1 KD mutations, and ErbB4 KD are available from the Protein Data Bank. Crystal structures of complexes with erlotinib, lapatinib, gefitinib, and AEE788, representing both active and inactive states of the kinase domain, were superimposed and inspected using the graphics program PyMOL.
Supporting Information Figure S1 Colony formation by early passage NMuMg cells stably expressing ERBB2 mutants. 2.56104 cells per well were plated in a six well plate and analyzed for colony formation. NMuMg cell line infected with MiGR1 vector is used as a control. Figure S2 Colony formation by late passage NMuMg cells. Late passage NMuMg cells stably expressing ERBB2 mutants were analyzed for colony formation. Cells infected with MiGR1 vector is shown as control. Figure S3 Structures of reversible and irreversible inhibitors used in this study. Figure S4 Cell based screen for lapatinib resistance. Schematic representation of lapatinb resistance screen performed with Ba/F3 cells stably expressing wild type ERBB2 kinase.
Residues affected by lapatinib resistance mutations identified in the in vitro screen were conserved in other ERBB members except ERBB3. Figure S5 Surface representation of EGFR kinase. Surface representation of EGFR, showing potential binding surfaces attributable to residues that differ between EGFR and ERBB2. Only one site is within the ATP binding pocket. A second is close by, Phe795 in EGFR is replaced by Tyr803 in ErbB2 visible near an ether chain of the inhibitor to the left of the binding cleft. Table S1 Representation of previously reported EGFR mutations homologous to ERBB2 mutants that were analyzed in this study. Table S2 Summary of relative resistance profiles of ERBB2 mutants against AEE 788, CL 387785 and WZ 4002 compared to lapatinib. Approximate fold increase in IC50 value of indicated ERBB2 mutant compared to wild type ERBB2 are calculated and classified as less, moderate or highly resistant. Author Contributions Conceived and designed the experiments: RKK RAE JD. Performed the experiments: RKK NB R