Method Details Plasmids and antibodies

The pLPCX retroviral vector (Clontech, CA) expressing full length HER2 with an N-terminal herpes simplex glycoprotein D (gD) tag was constructed and used for site directed mutagenesis. A retroviral vector, pRetro-IRES-GFP (Jaiswal et al., 2013), was used to stably express N-terminally FLAG-tagged HER2 WT.

Antibodies that recognize pHER2 (Y1221/22), pERK1/2, total ERK, HER2 and βactin were obtained from Cell Signaling Technology, MA. Anti-gD antibody (Genentech Inc., CA), anti-FLAG-M2 (Sigma, MO), and HRP-conjugated secondary antibodies (Pierce Biotechnology, IL) were also used in the study.

HER2 saturation scanning mutagenesis library was generated by replacing each residue in the TMD/JMD and flanking region (amino acids G641-K684) with possible 19 amino acids (836 possible single site mutants). Full length HER2 with an N-terminal gD (herpex simplex glycoprotein D) tag cloned into pLPCX retroviral vector (Clontech) was used as a plasmid template to generate mutants. The 836 mutagenic oligonucleotides and amplification primers needed to amplify the mutant primers were purchased from Agilent, CA (custom Quikchange HT protein engineering system™). The mutagenic oligos were amplified using a pair of flanking primers and used to mutagenize gDtagged HER2 cloned into the pLPCX retroviral vector. The mutant HER2 constructs were then transformed into Solopack Gold supercompetent E. coli cells (Agilent) and amplified. The retroviral vector DNA carrying the mutant HER2 pool was sequenced on Hiseq2500 (Illumina) to assess the mutation prevalence. Control mutant constructs carrying mutations were generated by site-directed mutagenesis and used as spike-in controls prior to generation of the retrovirus pool.

The retroviral plasmid library was transfected using Fugene6 (Roche, CA) into four 10 cm tissue culture dishes containing 0.25 × 10⁶ phoenix amphoteric packaging cells. BaF3 parental cells or those expressing Flag tagged WT HER2 were infected with the retroviral pool at a multiplicity of infection (MOI) of ~1 per cell using spinfection at 1,800rpm for 45 min, as previously described (Jaiswal et al., 2013). Pool of BaF3 stables cells was derived by culturing the cells in media supplemented with 1 μg/ml of puromycin for 14 days. Stable cells were washed twice with PBS and plated in quadruplicates in RPMI media devoid of IL-3. Surviving cells were collected at day 4 and genomic DNA was prepared (DNeasy Blood and Tissue Kit, Qiagen) and used to amplify the HER2 TMD/JMD regions. A 243–249bp amplicon was prepared from each replicate using a pool of three staggered primer pairs, F1 (5’–CTTGCCCCATCAACTGCAC 3’), F2 (5’-CCTTGCCCCATCAACTGCAC-3’), F3 (5’AGCCTTGCCCCATCAACTGCAC-3’), R1 (5’-TCGCTCCGCTAGGTGTCAGCGGCT-3’); R2 (5’-ATCGCTCCGCTAGGTGTCAGCGGCT-3’) and R3 (5’-GCATCGCTCCGCTAGGTGTCAGCGGCT-3’) and 50ng of genomic DNA using standard PCR conditions. Amplicons were column purified, quantified on Qubit fluorometer, pooled and used to construct libraries (NuGen Ovation Library System (NuGen) cat# 9092–256) with 75ng of DNA as the input. The libraries were then sequenced on HiSeq 2500 (Illumina) to generate 2 × 75-bp paired-end data. The reads were aligned to the HER2 sequence (Ensembl ID: ENST00000269571) using BWAMEM (version 0.7.10; http://arxiv.org/abs/1303.3997) with default parameters. For each mutant, the reads observed were counted using only high-quality bases (Q score > 30) from high-quality alignments (mapping quality >30). The frequency of each mutant was calculated as the number of observed mutant-reads divided by the total number of reads at that locus. The mutant allele-frequency at day 4 was used to estimate the enrichment of mutants in the pool.

BaF3 cells stably expressing HER2 mutants were washed twice with PBS and plated in 96-well plates (10,000 cells/well) in replicates of 12 in complete RPMI medium without IL-3. Cell viability was measured using the Cell Titer Glo Luminescence Cell Viability Kit (Promega), and plates were read on a Synergy 2 (Biotek Instruments) luminescence plate reader. Relative survival reported in cell survival studies is a ratio of relative luciferase activity (RLU) at day 4 over RLU measured at the time (day 0) the experiment was initiated.

HER2 expression levels were quantified by incubating cells with saturating amounts of an anti-HER2-PE antibody (BioLegend), washed, and read by flow cytometry (FACS Calibur) to determine the mean fluorescence intensities (MFI).

BaF3 cells stably expressing HER2 mutants were starved of IL-3 for 24 hours. Cells bearing HER2 mutants without and with WT HER2 were lysed at 1 × 10⁶ and 0.25 × 10⁶ cells per 30 μl Tris lysis buffer, respectively, supplemented with phosphatase inhibitors II and III (Sigma-Aldrich), and protease inhibitor (Roche). Lysates were centrifuged 5 min at 14,000 rpm to remove cell debris and 25 μl per well added to plates coated with antibodies to HER2 p-Tyr1248 and total HER2. Bound HER2 was detected with a Sulfo-Tag-labeled secondary antibody. Percent p-HER2 was calculated as follows: ((2 x Phospho-signal) / (Phospho-signal + Total signal)) x 100.

Western blots were performed as previously described (Jaiswal et al., 2013). For Western blot studies, BaF3 cells were grown in absence of IL-3 for 18 h. MCF10A cells were cultured in normal growth media containing 5% horse serum and EGF.

The input models were built using a truncated form (residues 649 – 679) of the median model reported in the NMR ensemble of a structure determined for wild-type HER2 dimer (PDBID: 2JWA) as our template. The orientation of the TMD dimer in the membrane was predicted using the OPM ppm server (Lomize et al., 2012). The proteinbilayer system was built by combining the oriented protein coordinates with a 60 × 60 membrane slab of POPC lipids and solvated with 15 Å of water containing 0.15 M NaCl on each side of the bilayer, removing overlapping lipids. Each system contained approximately 20,500 atoms. Langevin dynamics simulations were run under NPT conditions with a Berendsen barostat and Langevin thermostat (Berendsen et al., 1984) using the CHARMM36 force field (Huang and MacKerell, 2013) and ACEMD (Harvey et al., 2009) with a 4 fs time step using the hydrogen mass repartitioning scheme (Feenstra et al., 1999). Model equilibration for simulations of WT/WT, G660D/G660D, G660R/G660R, and WT/R678Q HER2 TMD dimers was conducted for 10 ns with simple harmonic positional constraints of backbone Cα atoms with a Langevin damping constant of 1.0 ps⁻¹. Constraints were then lifted for 90 ns production simulations, with Langevin damping constant of 0.1 ps⁻¹. A 400 fs Berendsen barostat relaxation time was used throughout. The stability of the final model was evaluated by calculating the backbone Cα root mean square deviation (RMSD) between the simulation at each time step and either the input model or the final model obtained at the end of the simulation. To check the robustness of the observed conformation in the G660D HER2 TMD homodimer, we repeated the simulation an additional 4 times and calculated pairwise RMSD values for each final state observed to assess the level of convergence between simulations. In all G660D mutant simulations, the side chain of the aspartate residues was protonated.

BaF3 cells stably expressing HER2 mutants either with or without WT HER2 were plated in 96 well plates in 100 μl of RPMI lacking IL-3. The cells were then treated with indicated concentrations of trastuzumab, pertuzumab, cetuximab, anti-ERBB3 antibodies (ERBB3.1(YW55.87.5) and ERBB3.2 (YW57.88.5 )) or Fab fractions of pertuzumab antibody or small molecule inhibitors. The Fab fraction of the pertuzumab antibody was generated and purified using papain, a non-specific thiol-enodpeptidase, immobilized on agarose resin (Pierce Fab preparation kit, Thermo Scientific).

Viable cell number was assessed 4 days after treatment using Cell Titer-Glo Luminescent cell viability assay kit (Promega Corporation) as described earlier (Jaiswal et al., 2013). Non-linear regression plot of antibodies and their fractions or of inhibitors were generated. Calculation of IC₅₀ was performed using GraphPad Prism 5.00 software. Data are presented as mean ± SEM of at least 3 replicates of a representative experiment that was repeated at least three times.

MCF10A cells stably expressing WT HER2 or mutant HER2 either alone or in combination with WT HER2 were seeded on growth factor reduced Matrigel (BD Biosciences) in the absence of any exogenous EGF or growth factor in a 8-well chamber slide as previously described (Jaiswal et al., 2013). Acini morphology was photographed on day 12 using EVOS microscope (Thermo Fischer) with a 10x objective.

About 20,000 BaF3 cells stably expressing either WT HER2 or G660D HER2 were plated on IL-3 free methylcellulose (Stemcell Technologies) in the absence or presence of various HER2 inhibitors and assessed for colony formation as previously described (Jaiswal et al., 2013). The plates were incubated at 37°C for 2 weeks and colonies were photographed using Gel count Imager (Oxford Optronix Ltd, UK).

BaF3 in vivo survival studies using cells expressing HER2 mutants were performed as previously described (Jaiswal et al., 2013). Briefly, 2 × 10⁶ BaF3 cells expressing either empty vector (EV), WT HER2 or mutant HER2 were implanted into 812 week old Balb/C nude mice by tail vein injection and followed for survival of the mice. For in vivo antibody efficacy study, 10 mg/kg anti-Ragweed antibody (control) or 10 mg/kg trastuzumab was administered intra-peritoneal once a week starting at 4 days post cell implantation. Each arm in the study contained ten mice. All animal studies were conducted under protocols approved by Genentech’s Institutional Animal Care and Use Committee (IACUC) guidelines.

In this study we analyzed blood samples from three patients (III.1, III.2 and III.3) and tumor DNA from one patient (III.3; Figure 1A)). The study was conducted with IRB approval and written patient informed consent.

Exome capture was performed using the Agilent SureSelect Human All Exome kit (50 Mb). Exome capture libraries were sequenced on HiSeq 2500 (Illumina) to generate 2 × 75 bp paired-end data. A targeted mean coverage of 88x with 94% bases covered at ≥10x was achieved for exome libraries.

All sequencing reads were evaluated for quality using the Bioconductor ShortRead package (Morgan et al., 2009). An all-against-all sample comparison was done on germline variants to confirm the patient matched tumor-normal pairing prior to additional data analysis.

Whole exome sequencing data were processed using the Genome Analysis Toolkit (GATK) (version v3.5–0-g36282e4) following best practices recommendations (DePristo et al., 2011; Van der Auwera et al., 2013). Reads were mapped to the human reference genome GRCh37 using BWA-MEM (version 0.7.10; http://arxiv.org/abs/1303.3997). Duplicate alignments were marked and removed using Picard tool (version 1.126) (http://broadinstitute.github.io/picard/) followed by indel realignment and base quality score recalibration. Haplotype Caller was used to generate gVCFs for all samples. Joint variant calling was performed for the 3 samples using GATK Genotype GVCFs. Variant quality score recalibration was carried out to estimate the confidence of variants called in the discovery cohort. Variant annotation was carried out using SnpEff (version 4.2) (Cingolani et al., 2012). Additional variant filtering and interpretation was done using Qiagen’s Ingenuity Variant Analysis software (www.qiagen.com/ingenuity). A set of final variants were manually reviewed using Integrative Genomics Viewer (IGV) (Robinson et al., 2011).

Samples were processed in one of two broad protocols applicable to solid tumors or hematologic cancers as previously described (Frampton et al., 2013). Samples were submitted to a Clinical Laboratory Improvement Amendment (CLIA)-certified, New York State-accredited, and CAP-accredited laboratory (Foundation Medicine) for hybrid capture followed by next-generation sequencing (NGS). All samples that advanced to DNA extraction contained a minimum of 20% tumor cells. DNA was extracted from formalin fixed paraffin embedded 10-μm sections. Adaptor-ligated DNA underwent hybrid capture for all coding exons of 287 or 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >500x using Illumina HiSeq sequencing technology.

Quantification and statistical analysis were done using R software. Comparison of relative cell survival between HER2 mutant and WT was performed using two-tailed Student t-test. Resulting p values were corrected using Bonferroni method. Corrected p value of 0.05 was used as significance threshold. Error bars (as shown in Figure 3) represent standard deviation of relative cell survival.