Article Figures & Data
Figures
- Fig. 1.
Schematic of the in vitro screen for ErbB2 drug resistance–associated mutations. An ErbB2 V659E expression construct was subjected to random mutagenesis. Cells expressing mutated ErbB2 that survived and grew in the presence of lapatinib or EXEL-7647 concentrations that prevented growth of cells expressing unmutagenized ErbB2 were selected and resistance-associated mutations were identified by DNA sequencing. The ErbB2 resistance-associated mutations were cloned and expressed in Ba/F3 cells to confirm that drug resistance was conferred by the detected mutation. Following stable expression of the mutated ErbB2 protein, survival IC50 values for all mutations and phosphorylation IC50 values for selected mutations were determined from 10-point dose-response assays.
- Fig. 2.
A, a schematic of the ErbB2 juxtamembrane and kinase domains shows the resistance-associated mutations identified in our studies. Functional domains are indicated. Seventeen different single amino acid substitutions, which map to 16 ErbB2 amino acid residues, are marked at the corresponding sequence positions. L785F was identified in both lapatinib and EXEL-7647 screens. B, amino acids where resistance-associated substitution mutations were identified are shown on an ErbB2 structural model. The model shown is an inactive form of the ErbB2 kinase domain with lapatinib docked in the ATP-binding site. The box shows a magnification of the ATP-binding region. Blue, activation loop; cyan, Cα-helix; green, nucleotide-binding loop; red, NH2-terminal extension; pink, COOH-terminal extension.
- Fig. 3.
Expression of mutant ErbB2 in Ba/F3 cells and determination of phosphorylation IC50 values. Ba/F3 cells selected for stable expression of resistance-associated mutations were used for ErbB2 and ERK phosphorylation IC50 determination. Cells were transferred to medium without serum or IL-3 2 h before compound addition. Following 1-h compound treatment (30,000, 10,000, 3,300, 1,100, 370, 123, 41, 14, 5, and 0 nmol/L), cells were harvested for Western blot analysis of pErbB2, total ErbB2 (FLAG), pERK, and total ERK. IC50 values were determined from a 10-point lapatinib or EXEL-7647 dose response. Western blot images of pErbB2, ErbB2, and pERK from cells expressing ErbB2 V659E, ErbB2 V659E_T798I, ErbB2 V659E_L785F, or ErbB2 V659E_L755S. IC50 values for pErbB2 were calculated using pErbB2 intensity after normalization to total ErbB2 levels. IC50 values for pERK were calculated using pERK intensity after normalization to total actin levels (data not shown). At least two immunoblot experiments were completed, and representative data are shown. The decreased ErbB2 protein level observed with 30 μmol/L EXEL-7647 treatments was associated with cell death (this effect was diminished in some samples by normalized protein loading).
- Fig. 4.
Lapatinib binds an inactive ErbB2 conformation and mutations that introduce steric interference or promote stability of the active kinase conformation can cause resistance. A, lapatinib is shown docked into inactive (left) and active (right) conformations of the ErbB2 structural model. In the active conformation, the Cα-helix (cyan) Glu-Lys salt bridge is formed, and the Cα-helix is positioned closer to the ATP-binding pocket where it clashes with lapatinib. Blue, activation loop. B, resistance-associated mutations predicted to cause lapatinib resistance by steric interference with lapatinib binding are shown. The amino acid mutation replaces a WT side chain in proximity to the ligand binding area with a bulkier side chain that interferes with lapatinib binding in the structural model.
- Fig. 5.
ErbB2 drug resistance–associated mutations are transforming in NIH-3T3 cells. Mutations identified by DNA sequencing in lapatinib-resistant cells were introduced into WT ErbB2 expression constructs by site-directed mutagenesis to resynthesize the resistance-associated mutations in the absence of the V659E primary mutation. The mutant ErbB2 constructs were expressed in NIH-3T3 cells and stable expressing cells were selected. Cells were grown to confluence. Growth mode was documented by observation and photography. A, uninfected NIH-3T3 cells grow to confluence and form a monolayer. B, NIH-3T3 cells expressing ErbB2 V659E fail to reach confluence and form foci of piled-up cells. C, NIH-3T3 cells expressing WT ErbB2 grow to confluence and form a monolayer as observed for uninfected cells. D, NIH-3T3 cells expressing ErbB2 L755S fail to reach confluence and form foci of piled-up cells as observed for ErbB2 V659E.
Tables
- Table 1.
ErbB2 mutations associated with lapatinib resistance and survival IC50 analysis of cloned ErbB2 resistance-associated mutations expressed in Ba/F3 cells
ErbB2 mutation Clones Mutation frequency (%) Protein domain Ba/F3 cell line Lapatinib EXEL-7647 IC50 (nmol/L) SD IC50 (nmol/L) SD None NA NA NA V659E 96 21 148 25 C630Y 1 1.1 EC — — — — — E717K 1 1.1 JM V659E_E717K 461 50 294 103 E719K 1 1.1 JM — — — — — E719G 1 1.1 JM V659E_E719G 894 162 43 6 L726F 8 9.0 Kinase V659E_L726F 1,056 65 315 88 T733I 2 2.2 Kinase V659E_T733I 670 25 515 153 L755S 5 5.6 Kinase V659E_L755S 2,861 746 371 13 P780L 4 4.5 Kinase V659E_P780L 1,127 76 448 129 S783P 5 5.6 Kinase V659E_S783P 3,273 5 181 61 L785F 15 16.9 Kinase V659E_L785F 1,154 65 554 175 T798I 35 39.3 Kinase V659E_T798I 3,339 289 419 131 Y803N 1 1.1 Kinase V659E_Y803N 496 170 123 24 E812K 6 6.7 Kinase V659E_E812K 407 74 187 1 D821N 1 1.1 Kinase V659E_D821N 249 5 318 49 V839G 1 1.1 Kinase V659E_V839G 286 14 96 8 L915M 1 1.1 Kinase V659E_L915M 253 53 152 89 S1002R 1 1.1 CT — — — — — NOTE: Mutations identified by DNA sequencing in lapatinib-resistant cells were introduced into ErbB2 V659E expression constructs by site-directed mutagenesis to resynthesize the resistance-associated mutations. The mutant ErbB2 constructs were expressed in Ba/F3 cells, and stable expressing cells were selected. Cell viability was measured following 48-h compound treatment in the absence of IL-3. Viability IC50 values were calculated from a 10-point lapatinib or EXEL-7647 dose response.
Abbreviations: EC, extracellular; JM, juxtamembrane; CT, COOH terminus.
- Table 2.
IC50 values for cell viability, ErbB2 phosphorylation, and ERK phosphorylation
Ba/F3 cell line Resistance mutation Survival IC50 (nmol/L) pErbB2 IC50 (nmol/L) pERK IC50 (nmol/L) Lapatinib EXEL-7647 Lapatinib EXEL-7647 Lapatinib EXEL-7647 V659E None 96 148 742 983 1,259 559 V659E_L755S L755S 2,861 371 3,610 954 9,375 1,248 V659E_L785F L785F 1,154 554 3,918 3,341 10,107 5,966 V659E_T798I T798I 3,339 419 >30,000 3,267 >30,000 3,296
Volume 14, Issue 8
