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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 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 adenocarci- noma (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 mu- tation 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), con- sistent 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.
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