Functional Analysis of Receptor Tyrosine Kinase Mutations in Lung Cancer Identiﬁes Oncogenic Extracellular Domain Mutations of ERBB2

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Functional analysis of receptor tyrosine kinase mutations in lung cancer identiﬁes oncogenic extracellular domain mutations of ERBB2

Heidi Greulicha,b,c,d,1, Bethany Kaplana,d, Philipp Mertinsd, Tzu-Hsiu Chend, Kumiko E. Tanakaa,d, Cai-Hong Yune, Xiaohong Zhanga, Se-Hoon Leea, Jeonghee Choa, Lauren Ambrogiod, Rachel Liaoa,d, Marcin Imielinskia,d, Shantanu Banerjia,d, Alice H. Bergera,d, Michael S. Lawrenced, Jinghui Zhangf, Nam H. Phoa,d, Sarah R. Walkera, Wendy Wincklerd, Gad Getzd, David Franka, William C. Hahna,b,d,g, Michael J. Eckh, D. R. Manid, Jacob D. Jaffed, Steven A. Carrd, Kwok-Kin Wonga,b,c, and Matthew Meyersona,d,g,i,j

aDepartment of Medical Oncology, gCenter for Cancer Genome Discovery, and hCancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115; Departments of bMedicine and iPathology, Brigham and Women’s Hospital, Boston, MA 02115; Department of cMedicine and jPathology, Harvard Medical School, Boston, MA 02115; dBroad Institute of Harvard and MIT, Cambridge, MA 02142; eDepartment of Biophysics, Peking University Health Science Center, Beijing 100191, China; and fDepartments of Biotechnology and Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited by William Pao, Vanderbilt–Ingram Cancer Center, Nashville, TN, and accepted by the Editorial Board July 24, 2012 (received for review February 23, 2012)

We assessed somatic alleles of six receptor tyrosine kinase genes Although most receptor tyrosine kinase mutations tested failed mutated in lung adenocarcinoma for oncogenic activity. Five of to score, novel extracellular domain mutations of ERBB2 were these genes failed to score in transformation assays; however, novel oncogenic. Our results indicate a unique therapeutic opportunity recurring extracellular domain mutations of the receptor tyrosine for patients with lung and breast cancer who harbor extracellular kinase gene ERBB2 were potently oncogenic. These ERBB2 extra- domain mutations of ERBB2. cellular domain mutants were activated by two distinct mecha- Results nisms, characterized by elevated C-terminal tail phosphorylation ERBB2 CELL BIOLOGY or by covalent dimerization mediated by intermolecular disulfide Extracellular Domain Mutations of Found in Cancer are bond formation. These distinct mechanisms of receptor activation Oncogenic. In the most comprehensive lung adenocarcinoma tar- converged upon tyrosine phosphorylation of cellular proteins, geted sequencing experiment thus far, 623 genes were sequenced in 188 lung adenocarcinomas, identifying 1,013 nonsynonymous so- impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 fi independence by the ERBB2 extracellular domain mutants was matic mutations and 26 signi cantly mutated genes (12). In addition abrogated by treatment with small-molecule inhibitors of ERBB2, to mutated genes already well characterized in lung adenocarcinoma (13), the significant genes included known tumor suppressors raising the possibility that patients harboring such mutations fi and several receptor tyrosine kinases, putative but unproven could bene t from ERBB2-directed therapy. oncogenes. In an effort to determine whether these uncharacterized receptor tyrosine kinase mutations are oncogenic, we analyzed the HER2 | breast cancer | bladder cancer four most significantly mutated receptor tyrosine kinase genes identified by multiple statistical methods, EPHA3, ERBB4, FGFR4, ung cancer is the leading cause of cancer death, accounting and NTRK3, and two genes that failed to achieve statistical signif- Lfor over 150,000 deaths annually in the United States alone icance, NTRK2 and ERBB2, due to a cluster of mutations in the (1). Current treatment options are thus inadequate for the majority kinase domain of NTRK2 and an extracellular domain mutation of patients and additional therapies are needed. Mutationally ac- of unknown significance in ERBB2 (Fig. S1). We expressed the tivated oncogenes that promote tumorigenesis represent poten- mutant alleles in NIH 3T3 cells and examined oncogenic activity in tial drug targets due to frequent dependency of tumor cells on soft agar assays. such oncogenes (2, 3), and somatically altered receptor tyrosine None of the somatic alleles of EPHA3, ERBB4, FGFR4, NTRK2, kinases in particular have been successfully exploited as thera- or NTRK3 were found to support anchorage-independent pro- peutic targets in several cancers. liferation in soft agar assays (Figs. S1 and S2A). In contrast, ectopic The prototypical therapy targeted to a somatically activated expression of FGFR4 V550E, recurrent in rhabdomyosarcoma and tyrosine kinase oncogene is imatinib mesylate, which targets the oncogenic in NIH 3T3 cells (14), and FGFR4 K645E, modeled after BCR-ABL fusion protein in chronic myelogenous leukemia (4). the activating FGFR3 K650E mutation found in multiple can- Targeted therapies developed for lung cancer include gefitinib cers (15), resulted in soft agar colony formation (Fig. S2A). and erlotinib, small-molecule inhibitors of mutationally activated Moreover, we could not detect EPHA3 protein expression in EGFR in lung adenocarcinoma (5–8), and crizotinib, a small- three lung cancer cell lines harboring EPHA3 mutations (Fig. molecule inhibitor of the EML4-ALK translocation product in S2B). Somatic mutations of EPHA3, ERBB4, FGFR4, NTRK2, and NTRK3 reported in lung adenocarcinoma thus do not confer lung adenocarcinoma (9). Trastuzumab, a monoclonal antibody phenotypes expected of receptor tyrosine kinase oncogenes. inhibitor targeting ERBB2, and the small-molecule EGFR/ ERBB2 inhibitor lapatinib are effective in ERBB2-amplified patients with breast cancer (10, 11). Theadventofnext-generationsequencing technologies has Author contributions: H.G., P.M., S.R.W., W.W., D.F., J.D.J., S.A.C., and K.-K.W. designed research; H.G., B.K., P.M., T.-H.C., K.E.T., S.-H.L., L.A., R.L., and W.W. performed research; enabled compilation of large somatic mutation datasets from M.S.L. and G.G. contributed new reagents/analytic tools; H.G., P.M., C.-H.Y., X.Z., J.C., M.I., cancer sequencing studies. Statistical methods that examine S.B., A.H.B., M.S.L., J.Z., N.H.P., W.W., G.G., D.F., W.C.H., M.J.E., D.M., K.-K.W., and M.M. differences in gene mutation frequency can reveal evidence of analyzed data; and H.G. and M.M. wrote the paper. positive selection; however, demonstration of the contribution of The authors declare no conflict of interest. a mutated gene to tumorigenesis additionally requires functional This article is a PNAS Direct Submission. W.P. is a guest editor invited by the Editorial validation. To identify new lung cancer oncogenes, we system- Board. atically assessed somatic alleles of significantly mutated receptor 1To whom correspondence should be addressed. E-mail: [email protected]. tyrosine kinase genes reported in patients with lung adenocar- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cinoma (12) for activity in cellular transformation assays. 1073/pnas.1203201109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203201109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Of the four mutations reported in ERBB2, S310F and A A775_G776insYVMA (“insYVMA”) are predicted to encode the full-length protein (Fig. S1). Whereas the insYVMA mutation of the kinase domain of ERBB2 is already well characterized (16, 17), mutations of the extracellular domain have not been pBp wt G309E S310F S310Y functionally analyzed. We therefore focused on the S310F mutation in exon 8 of ERBB2, found in 1/188 lung adenocarcinoma B wt G309E S310F insYVMA pBp samples (12). Additional reports of extracellular domain muta- S310Y D845A insYVMA D845A ERBB2 tions of ERBB2 included G309E in 1/183 breast cancer samples Actin and S310Y in 1/63 squamous lung cancer samples (18), S310F in C 100 2/112 breast cancers (19), 1 S310F and 1 S310Y in 258 lung AALE 80 D adenocarcinomas sequenced by the Cancer Genome Atlas Net- 60 work (Fig. S3 A and B), S310F in 1/65 breast cancers (20), and wt G309E S310F pBp 40 insYVMA S310Y D845A S310F in 1/316 ovarian cancers (21). An S310F mutation was 20 ERBB2

also found in a bladder cancer cell line, 5637 (22). Colonies of Number 0 Vinculin wt

We examined genomic data for samples with extracellular do- pBp S310F S310Y G309E main mutations of ERBB2. One breast cancer sample harbored D845A an additional kinase domain mutation of ERBB2, L755S, and one E insYVMA lung cancer sample harbored a mutation of KRAS,G12F(Fig. S3C); none had mutations of EGFR. One breast cancer sample exhibited high-level amplification of ERBB2 in the genome, whereas pBp wt L49H T216S C311R the other samples did not (Fig. S3C). Two of four patients with lung cancer were former smokers. However, given the small number of samples analyzed, we lack power to determine whether there are any systematic associations of ERBB2 extracellular domain N319D E321G D326G C334S V750E mutations with the presence or absence of other known driver mutations, ERBB2 amplification, or smoking status. NIH 3T3 cells overexpressing wild-type ERBB2 exhibited a V777A insYVMA insV D845A weak anchorage-independent phenotype (Fig. 1 A and B), consistent with previous reports (23). In contrast, the G309E, S310F, F and S310Y mutants supported robust colony formation in soft agar N319D insV E321G D326G D845A wt L49H C311R C334S V750E V777A insYVMA pBp T216S (Fig. 1 A and B), similar to an ERBB2 kinase domain insertion ERBB2 – mutant (16, 24 26). A kinase-inactive mutant, D845A, failed to Phospho-ERBB2 form any colonies. AALE human lung epithelial cells were simi- Y1221/1222 larly transformed to anchorage independence by the extracellular Actin mutants of ERBB2 (Fig. 1 C and D). We have thus identified oncogenic somatic mutations of the extracellular domain of Fig. 1. Extracellular domain mutations of ERBB2 found in lung and breast ERBB2 in lung and breast cancer, occurring at a rate of about 1%, cancer are oncogenic. (A) NIH 3T3 cells expressing ERBB2 extracellular approximately half that of the ERBB2 kinase domain mutations mutants were assessed for colony formation in soft agar. (B) Anti-ERBB2 immunoblot on NIH 3T3 lysates. (C) AALE human airway epithelial cells previously described in lung adenocarcinoma (12, 24, 26). fi expressing ERBB2 extracellular mutants also exhibited an increase in soft ERBB2 was reported to be a signi cantly mutated gene in agar colony formation. (D) Anti-ERBB2 immunoblot on AALE lysates. (E)NIH glioblastoma (27). Paradoxically, only three of the reported 3T3 cells expressing ERBB2 mutants reported in glioblastoma were assessed mutations, C311R, E321G, and C334S, were transforming (Fig. 1 for colony formation in soft agar. (F) Immunoblot analysis of ERBB2 protein E and F). Upon closer inspection, it became apparent that all 7 and phosphorylation state on lysates of NIH 3T3 expressing ERBB2 mutations glioblastoma samples harboring ERBB2 mutations were from reported in glioblastoma. pBp, pBabe puro vector; insYVMA, A775_G776- a single TCGA sample batch. Because the reported data were insYVMA; insV, ERBB2 A775_G776insV/G776C; WT, wild-type ERBB2. derived from 91 samples in four sample batches, it was unlikely that all 7 samples would be in the same sample batch by chance (Fisher’s exact P = 0.000002). Nor could prior patient treatment in vitro transforming activity, C311R and C334S, affect cysteine account for this cluster of ERBB2-mutated samples. This analysis residues, we examined the crystal structure of ERBB2 (29) to ask raised the possibility that the reported mutations were artifacts whether these changes affect disulfide bonds. Both C311 and C334 of whole-genome amplification, as the mutations in the TCGA are involved in disulfide bond formation, with C299 and C338, study were not validated in unamplified DNA. respectively (Fig. 2A). These intramolecular disulfide bonds are Sequenom genotyping of the original unamplified glioblas- presumably disrupted in the C311R and C334S mutants. toma DNA samples, corresponding to the whole-genome am- We hypothesized that disruption of intramolecular disulfide plified material in which the ERBB2 mutations were discovered, bonds might result in formation of intermolecular disulfide bonds failed to detect the reported ERBB2 mutations (Fig. S4A), by the remaining unpaired cysteines. We tested this hypothesis by whereas most mutations reported in other significantly mutated running lysates on nonreducing and reducing gels in parallel. genes in this sample batch were present in the unamplified DNA Whereas wild-type ERBB2 showed no evidence of dimerization (Fig. S4B). Consistent with the possibility that these ERBB2 under these conditions, C311R and C334S formed high-molecular- mutations are artifacts, no mutations of ERBB2 were reported in a weight species consistent with ERBB2 dimers on nonreducing parallel study (28). Because of these inconsistencies, we checked SDS/PAGE gels (Fig. 2B). E321G did as well, possibly due to unamplified tumor DNA for the three mutations in lung and disruption of salt bridges that E321 forms with K369 and R434 breast cancer originally detected in whole-genome amplified that stabilize the structure of the disulfide-bonded loops (Fig. 2 A material. All three mutations were confirmed in native DNA and B). In contrast, there was no evidence of dimerization by the (Figs. S3C and S4 C–F). transforming insYVMA kinase domain mutant (Fig. 2B). We then examined the mechanism of activation of the mutants ERBB2 Extracellular Domain Mutations Activate the Receptor by Two found in breast and lung cancer. The S310F mutant protein Distinct Mechanisms. The oncogenic mutations of the ERBB2 ex- was hyperphosphorylated, similar to the kinase domain mutant tracellular domain cluster in subdomain II, a region characterized insYVMA (Fig. 3A). However, the C-terminal tail of the G309E by 11 disulfide bonds (29). Because two ERBB2 mutants with mutant was not hyperphosphorylated (Fig. 3A), like that of other

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203201109 Greulich et al. Downloaded by guest on September 25, 2021 A ERBB2 S310F-expressing cells (Table 1) were also hyperphosphorylated in the G309E-expressing cells (Dataset S1). Fur- thermore, the 92 individual peptides phosphorylated twofold or higher in the ERBB2 S310F-expressing cells compared with ERBB2 wild-type–expressing cells exhibit a fold change distri- bution that is skewed toward the top of the list of hyperphosphorylated peptides in the G309E-expressing cells in a statistically signiﬁcant manner, with a rank-test P value of 2.2 × − 10 16 (SI Experimental Procedures). These data indicate that despite activation by distinct mechanisms, the two ERBB2 mutants use similar downstream effector pathways to transform cells. ERBB2 itself was hyperphosphorylated in S310F-expressing cells but not G309E-expressing cells, consistent with immunoblot data (Table 1, Dataset S1, and Fig. 3A). Interestingly, the EGFR/ ERBB2 inhibitor MIG6, encoded in human DNA by ERRFI1, was hyperphosphorylated in both the G309E- and S310F- expressing cells (Table 1 and Dataset S1), correlating with the previously described dependence of association with ERBB2 on ERBB2 activity but not C-terminal autophosphorylation (30). A number of proteins regulating cytoskeletal dynamics and

B Nonreducing Reducing cell motility were found to be prominently hyperphosphorylated in the ERBB2 S310F cells (Table 1), including the murine homo- logs of CRK, DLG1, CCD88A, IQGAP, and PEAK1, as well as components of the cytoskeleton (Table 1). Altered cell motility may wt wt C311R E321G C334S V777A insYVMA pBp C311R E321G C334S V777A insYVMA pBp D845A D845A Dimer thus be closely linked to the transformed phenotype measured by the soft agar assay. PTPN11, a phosphatase involved in activation ERBB2 Monomer of Erk proteins in response to growth factor stimulation and intriguingly required for growth and metastasis of HER2-positive breast cancer cells (31, 32), was also prominently phosphorylated CELL BIOLOGY Actin in the ERBB2 S310F cells. Of note, there was considerable overlap between the proteins phosphorylated in response to ERBB2 S310F Fig. 2. Oncogenic extracellular domain mutations of ERBB2 reported in glioblastoma cause disulfide bond remodeling. (A) Model of the ERBB2 di- expression and proteins reported to be phosphorylated in human mer made by superimposing the human [Protein Data Bank (PDB) ID code mammary epithelial cells in response to knockdown of PTPN12, 2A91] and rat (PDB ID code 1N8Y) ERBB2 extracellular domain crystal a negative regulator of EGFR and ERBB2 (33). structures onto an EGF-bound EGFR extracellular domain dimer crystal structure (PDB ID code 1IVO). Intramolecular disulfide bonds are indicated in Oncogenic Activity of ERBB2 Extracellular Domain Mutants Is Sensitive green. (B) Immunoblot analysis of ERBB2 extracellular mutants reported in to Treatment with ERBB2 Inhibitors. Introduction of a kinase-inacti- glioblastoma reveals formation of covalent dimers on nonreducing gels. vating D845A mutation into cDNAs harboring ERBB2 extracel- pBp, pBabe puro vector; WT, wild-type ERBB2. lular domain mutations prevented soft agar colony formation by transduced NIH 3T3 cells (Fig. S5B, Bottom Right). To facilitate inhibitor testing, we expressed the ERBB2 mutants in murine Ba/ fi mutants of ERBB2 that dimerized by intermolecular disul de F3 cells and derived IL-3 independent lines. Expression of the bonding (Figs. 1F and 3A). We therefore investigated the dimerization capacity of ERBB2 G309E and found that this mutant did indeed form reduction-sensitive dimers (Fig. 3B). There are six cysteine residues involved in three intramolecular A disulfide bonds in the region below the dimerization arm of S310F E321G wt pBp ERBB2; replacement of any of these six cysteines with serine G309E C311R insYVMA ERBB2 conferred the ability to form reduction-sensitive dimers and transform NIH 3T3 cells (Fig. S5 A and B). A decrease in C-terminal Phospho-ERBB2 phosphorylation was also observed on the ERBB2 cysteine

mutants despite a robust soft agar phenotype (Fig. S5C). Actin ERBB2 Extracellular Domain Mutants Effect Transformation Using B Common Downstream Machinery. We have thus deﬁned two distinct D845A wt S310F E321G pBp G309E S310Y mechanisms of activation of extracellular domain mutants of insYVMA the ERBB2 receptor tyrosine kinase: elevation of C-terminal ERBB2 phosphorylation and formation of disulﬁde-linked dimers. In Nonreducing order to determine whether these two classes of ERBB2 mutants

use similar pathways to effect oncogenic transformation, we used ERBB2 stable isotope labeling by amino acids in cell culture (SILAC) Reducing combined with immunoafﬁnity enrichment of tyrosine-phosphor- Actin ylated peptides to compare differences in global protein tyrosine Nonreducing phosphorylation using quantitative mass spectrometry. Whereas only a slight increase in phosphopeptide ratios was Fig. 3. ERBB2 mutants found in lung and breast cancer form reduction- seen in the ERBB2 G309E-expressing cells over wild type, the sensitive dimers that exhibit diminished C-terminal tail phosphorylation. (A) cells expressing ERBB2 S310F exhibited a more substantial in- Immunoblot analysis of ERBB2 protein and tyrosine 1221/1222 phosphory- crease in peptide phosphorylation (Fig. S6), correlating with the lation state on lysates of NIH 3T3 expressing ERBB2 extracellular domain greater oncogenic activity of S310F. Forty-four of 47 endogenous mutations. (B) Immunoblot analysis of ERBB2 dimers trapped by non- proteins with peptides phosphorylated twofold or higher in the reducing SDS/PAGE. pBp, pBabe puro vector; WT, wild-type ERBB2.

Greulich et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Table 1. Proteins containing peptides phosphorylated twofold the wild-type ERBB2 or the kinase domain mutant, insYVMA. or higher in cells expressing ERBB2 S310F than in cells expressing Importantly, the 95% confidence intervals of the IC50s for ex- wild-type ERBB2 implicate events that impact cell motility tracellular domain mutants S310F, S310Y, and E321G in response † to treatment with small-molecule inhibitors were generally lower Gene S310F/WT* No. phosphopeptides than the corresponding limits for wild-type ERBB2 or insYVMA CSNK1A1 83.5 1 (Fig. S7A). Inhibitor efficacy furthermore correlated with in- CRK 19.3 1 hibition of ERBB2 phosphorylation (Fig. S7 B–E). DLG3 17.1 1 The reversible inhibitor lapatinib was 5- to 10-fold less effective than neratinib and afatinib (Fig. 4D), perhaps due to the more AHNAK 14.8 2 fi DLG1 11.1 1 ef cient recovery of receptor activity, evidenced by increases in ERBB2 10.2 11 phospho-ERBB2 and phospho-Akt following inhibitor washout in CCDC88A 9.3 2 lapatinib-treated cells but not in neratinib-treated cells (Fig. S8). However, cells expressing the extracellular domain mutants were IQGAP1 9.3 1 significantly more sensitive to lapatinib than cells expressing ERRFI1 9.1 3 insYVMA (Fig. 4D). Trastuzumab treatment effectively inhibi- SEMA4C 8.0 1 ted survival of Ba/F3 cells expressing mutants of G309 and S310, PEAK1 7.0 3 but curiously had less of an effect on cells transformed by the STXBP3 7.0 1 other mutants (Fig. 4E). Although the cancer-derived mutations CAV1 6.4 3 are located in the same region of the receptor as the epitope PTPN11 6.2 1 bound by trastuzumab, these results indicate that mutations of ERBB2IP 5.6 4 G309 or S310 do not inhibit trastuzumab binding. ANXA1 5.4 2 We have previously shown that the lung cancer cell line NCI- TLN1 5.3 2 H1781, harboring an ERBB2 kinase domain mutation, is sensitive ANXA2 5.1 9 to treatment with the irreversible inhibitor afatinib (34). In DOK1 5.1 5 contrast, the endometrial cancer cell line AN3CA is character- G6PD 4.9 1 ized by FGFR2 mutation but not by ERBB2 mutation (35). Using TNK2 4.8 1 these two cancer cell lines as controls, we tested whether ERBB2 SH2B3 4.6 1 inhibition affected survival of a bladder cancer cell line, 5637, LAYN 4.4 2 harboring an ERBB2 S310F mutation (22). Whereas ERBB2 CFL1 4.3 2 inhibition alone was effective against the NCI-H1781 cells, SHC1 4.2 2 a combination of ERBB2 inhibition and Mek inhibition was ACTB 3.5 1 necessary for abrogation of 5637 cell survival with an IC50 comparable to that for the NCI-H1781 cells (Fig. 4F). Neither VIM 3.5 3 fi AXL 3.5 3 inhibitor had a signi cant effect on the AN3CA cells alone or in combination. These results suggest a possible treatment option for EPS8 3.0 1 patients with lung and breast cancer harboring these mutations. PLCG1 2.9 1 PABPC3 2.8 1 Discussion CALM1 2.7 2 We functionally analyzed mutated receptor tyrosine kinase genes LDLR 2.7 1 found in lung adenocarcinomas. None of the somatic alleles of CTNND1 2.6 1 EPHA3, ERBB4, FGFR4, NTRK3,orNTRK2 were found to be GAB1 2.6 1 oncogenic in NIH 3T3 cells. There are three possible explan- EEF1A2 2.5 1 ations for the lack of oncogenic transformation by these mutant CDK2 2.4 1 receptor tyrosine kinase genes. The reported significantly mu- VASP 2.2 1 tated genes may in fact be tumor suppressor genes, contributing RIN1 2.2 1 to tumorigenesis but not scoring in a transformation assay STAT3 2.2 2 designed to detect dominant gain-of-function oncogenes. The EEF1A1 2.2 1 absence of nonsense and frameshifting mutations of EPHA3, PIK3R2 2.2 1 ERBB4, NTRK2, and NTRK3 argues against a role in tumor CDK1 2.2 1 suppression, as all known significantly mutated tumor suppressor MAPK1 2.1 1 genes found in the lung adenocarcinoma study harbored muta- ABI1 2.1 1 tions resulting in premature termination. RPS27 2.1 2 A second explanation for the absence of oncogenic trans- RPS10 2.1 1 formation is that the tested somatic alleles are in fact gain-of- MYO9B 2.0 1 function and oncogenic, and we simply used the wrong assay to uncover an oncogenic phenotype. This argument is difficult to *Mean fold increase in phosphorylation of the most phosphorylated peptide refute; however, the ability of FGFR4 V550E and K645E and an for each protein. † ETV6-NTRK3 gene fusion (34) to support anchorage-independent Total number of distinct phosphopeptides detected for each protein. growth argues against such an explanation. Furthermore, we find it unlikely that three of three lung adenocarcinoma cell lines harboring EPHA3 mutations would fail to express detectable extracellular mutations of ERBB2 conferred IL-3 independence EPHA3 if such mutations were in fact oncogenic. fi more ef ciently than wild-type ERBB2, whereas the vector A third explanation for the absence of oncogenic trans- control and kinase-inactive form of ERBB2 were not able to formation is that the reported mutations are passenger muta- support IL-3–independent growth (Fig. 4A). tions, and more refined statistical methods are needed to detect Ba/F3 cells transformed with the ERBB2 extracellular domain evidence of positive selection. For example, as nonexpressed mutants were treated with the irreversible ERBB2 inhibitors genes may exhibit higher mutation rates than expressed genes neratinib and afatinib, resulting in effective abrogation of cell (37), incorporation of sample-specific gene expression data from survival, with IC50s in the low nanomolar range (Fig. 4 B and C). parallel RNA sequencing may assist in a more accurate estimation Cells expressing the extracellular domain mutants exhibited in- of gene-specific background mutation rates. Moreover, the effect creased sensitivity to these inhibitors relative to cells expressing of replication timing on mutation of individual genes (38, 39) was

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203201109 Greulich et al. Downloaded by guest on September 25, 2021 A B

Vec+IL-3 wild-type G309E S310F S310Y E321G insYVMA IC50 (µM) 0.7031 0.0023 0.0011 0.0013 0.0012 0.0020 0.0023 C D

Fig. 4. Ba/F3 cells transformed to IL-3 independence

Vec+IL-3 wild-type G309E S310F S310Y E321G insYVMA Vec+IL-3 wild-type G309E S310F S310Y E321G insYVMA with the ERBB2 extracellular domain mutants are IC50 (µM) 1.3090 0.0057 0.0032 0.0027 0.0029 0.0038 0.0054 IC50 (µM) 2.5260 0.0201 0.0107 0.0094 0.0106 0.0207 0.1755 sensitive to ERBB2 inhibition. (A) Proliferation of Ba/ F3 cells expressing mutant forms of ERBB2 upon IL-3 E F withdrawal. (B) Survival of ERBB2-transformed Ba/F3 cells in response to neratinib. (C) Survival of ERBB2- transformed Ba/F3 cells in response to afatinib. (D) Survival of ERBB2-transformed Ba/F3 cells in response to lapatinib. (E) Survival of ERBB2-transformed Ba/F3 cells in response to trastuzumab. (F) Response of

cancer cell lines NCI-H1781, AN3CA, and 5637 to a CELL BIOLOGY combination of Mek and ERBB2 inhibition. The concentration of Mek inhibitor PD184352, 1 μM,

Vec+IL-3 wild-type G309E S310F S310Y E321G insYVMA H1781 AN3CA 5637 H1781+PD AN3CA+PD 5637+PD was chosen for lack of an effect alone on survival IC50 (µg/ml) 0.2612 0.1563 0.0882 0.0508 0.0531 0.2955 0.2512 IC50 (µM) 0.220 14.480 2.032 0.112 3.128 0.215 of these cell lines.

not modeled into the background mutation rate calculation and The rat neu oncogene, an ERBB2 ortholog identified as the may similarly confound the results of significance testing. transforming agent in nitrosoethylurea-induced rat neuroblasto- In contrast to the other receptor tyrosine kinase mutants mas (42), harbors a mutation corresponding to V659E in the tested, an extracellular domain mutation of ERBB2 transformed transmembrane domain of human ERBB2 (43). Mutant neu but NIH 3T3 cells to anchorage independence. This result demon- not wild-type neu formed covalent high-molecular-weight species strates that infrequently mutated but genuine oncogenes can be under nonreducing conditions, consistent with disulfide bond- found in cancer sequencing data even if the genes fail to achieve mediated receptor dimerization (44). Mice expressing a mouse statistical significance. ERBB2 was reported as significantly mu- mammary tumor virus encoding wild-type neu developed mam- tated in glioblastoma (27) but the observed mutations could not be mary tumors from which spontaneously mutated forms of neu, confirmed in patient-matched unamplified tumor DNA, raising characterized by in-frame deletions in domain IV of the extra- the possibility that the reported mutations were artifacts of whole- cellular domain, could be isolated (45). These spontaneous de- genome amplification. In response to these data, the TCGA letion mutants, which typically removed a single cysteine residue, consortium is no longer to our knowledge performing any se- were oncogenic and migrated as dimers on nonreducing gels (46). quence analysis of whole-genome–amplified DNA. The availability of inhibitors effective against the extracellular We have identified a unique mechanism of activation of ERBB2 ERBB2 mutants present in lung, breast, ovarian, and bladder in tumor cells, namely, formation of covalent dimers linked by cancer raises therapeutic possibilities. The efficacy of a combi- intermolecular disulfide bonds in subdomain II. It is straightforward nation of ERBB2 and Mek inhibition on a bladder cancer cell to envision how the cysteine substitution mutants of the ERBB2 line harboring an ERBB2 S310F mutation further indicates the extracellular domain may lead to intermolecular disulfide bonding. clinical utility of this approach; however, it is not yet mechanis- G309 is located in close proximity to the C299-C311 disulfide bond; tically clear why both inhibitors are necessary for this effect, replacement of this compact residue with a bulkier residue such requiring further study. More broadly, our results suggest that as glutamate may prevent formation of this intracellular di- a clinical trial of ERBB2 inhibition, alone or in combination fi sul de bond, leaving unpaired cysteine residues available for in- with other agents, in patients with cancer harboring extracellular fi termolecular disul de bond formation. We furthermore speculate domain mutations of ERBB2 across tumor types is warranted. that the S310F and S310Y mutations may result in hydrophobic interactions between the aromatic rings of the newly introduced Experimental Procedures 310F or 310Y with Y274 and F279 of the neighboring molecule, Cell Culture. NIH 3T3 cells (ATCC) were maintained in DMEM (Cellgro) sup- promoting noncovalent dimerization and kinase activation. plemented with 10% (vol/vol) calf serum (Invitrogen). Ba/F3 cells were There is precedent in the literature for activation of receptor maintained in RPMI 1640 (Cellgro) with 10% FCS (Gemini Bioproducts) and tyrosine kinases by disulfide-mediated dimerization. Somatic and 10 ng/mL interleukin-3 (BD Biosciences). AALE cells were grown in SAGM germ-line mutations of the extracellular domain of the receptor media (Lonza). NCI-H1781 and 5637 cells were grown in RPMI 1640 with 10% tyrosine kinase FGFR3 that introduce unpaired cysteine residues FCS, and AN3CA cells were grown in MEM (Cellgro) with 10% FCS. have been described in bladder cancer and thanatophoric dysplasia, respectively (15). These mutations caused reduction-sensitive di- Retroviral Transduction. cDNAs were ectopically expressed in NIH 3T3, AALE, mer formation, activated FGFR3 kinase activity, and supported and Ba/F3 using a Gateway-modified pBabe puro vector. Details can be found anchorage-independent proliferation of NIH 3T3 cells (40, 41). in SI Experimental Procedures.

Greulich et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 Soft Agar Assay. Soft agar assays were performed as described in ref. 47. phosphatase inhibitors (Calbiochem). One aliquot was boiled in NuPage LDS 4× Briefly, 5 × 103 to 5 × 104 cells were suspended in media containing 0.33% sample buffer (Invitrogen) containing 100 mM DTT (final concentration of 20 Select agar (Invitrogen) and plated on a bottom layer of media containing mM DTT) and one aliquot was boiled in sample buffer without reducing agent. 0.5% Select agar in a six-well plate. Plates were incubated at 37 °C 2 wk Samples were run on 4–12% gradient polyacrylamide gels (Invitrogen). before imaging. AALE colonies were photographed at 5× magnification and one field was counted 2–3 wk after plating. SILAC Experiments. Experiments were performed as described in ref. 47. Full experimental details and statistical methods can be found in SI Inhibitor Assays. Totals of 2,000–4,000 Ba/F3 or 3,000 NCI-H1781, 5637, and Experimental Procedures. AN3CA cells were seeded in 96-well plates, incubated with the indicated concentrations of inhibitor for 3 d, and assessed for cell survival with the ACKNOWLEDGMENTS. We thank Dr. James Bradner for providing lapatinib, Dr. Jesse Boehm for supplying receptor tyrosine kinase cDNAs, Dr. Somase- WST-1 reagent (Roche). PD184352 (CI-1040) was obtained from Sigma. Afati- kar Seshagiri for genotyping of ERBB2 mutations found in ref. 18 in native nib, neratinib, and trastuzumab were purchased commercially, and lapatinib DNA, Dr. Emanuele Pelscandolo and Ms. Christina Go of the Dana-Farber was purified from patient-discarded tablets by James Bradner. Survival data Cancer Institute Center for Cancer Genome Discovery for genotyping the were analyzed using the Prism GraphPad software. ERBB2 mutation found in ref. 12 in native DNA, Drs. Angela Brooks and Andrew Cherniack for assistance with genomic data, and Drs. Hideo Wata- Dimerization Assay. The dimerization assay was performed as described in ref. nabe and Rameen Beroukhim for helpful discussions. H.G. is supported by fl a grant from Uniting Against Lung Cancer. This work was also supported in 46. Brie y, cells were washed twice with cold PBS containing 10 mM iodoace- part by National Cancer Institute Grants R01CA109038, R01CA116020, and tamide (Sigma) and lysed with 500 μL TGP buffer (50 mM Tris, pH 7.4, 1% Triton, P20CA90578 (to M.M.); the American Lung Association; the Seaman Foun- 10% glycerol, 10 mM iodoacetamide) containing protease inhibitors (Roche) and dation; and the Monopoli Foundation (M.M.).

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