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A Framework for Identification of Actionable Cancer Genome

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A Framework for Identification of Actionable Cancer Genome A framework for identification of actionable cancer genome dependencies in small cell lung cancer Martin L. Sosa,b,c,d,1,2, Felix Dietleina,b,1, Martin Peifera,b, Jakob Schöttlea,b, Hyatt Balke-Wanta,b, Christian Müllera,b, Mirjam Kokera,b, André Richterse,f, Stefanie Heyncka,b, Florian Malchersa,b, Johannes M. Heuckmanna,b, Danila Seidela,b, Patrick A. Eyersg, Roland T. Ullrichb, Andrey P. Antonchickh, Viktor V. Vintonyakh, Peter M. Schneideri, Takashi Ninomiyaj, Herbert Waldmanne,h, Reinhard Büttnerk, Daniel Rauhe,f, Lukas C. Heukampk, and Roman K. Thomasa,b,k,2 aDepartment of Translational Genomics, University of Cologne, 50931 Cologne, Germany; bMax Planck Institute for Neurological Research, 50931 Cologne, Germany; cHoward Hughes Medical Institute, dDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; eChemical Genomics Center of the Max Planck Society, 44227 Dortmund, Germany; fTechnical University Dortmund, D-44221 Dortmund, Germany; gYorkshire Cancer Research (YCR) Institute for Cancer Studies, Cancer Research United Kingdom (CR-UK)/YCR Sheffield Cancer Research Centre, Department of Oncology, University of Sheffield, Sheffield S10 2RX, United Kingdom; hMax Planck Institute of Molecular Physiology, D-44227 Dortmund, Germany; iInstitute of Forensic Medicine, University of Cologne, 50823 Cologne, Germany; jDepartment of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 700-8558 Okayama, Japan; and kInstitute of Pathology, University of Cologne, 50924 Cologne, Germany Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved September 11, 2012 (received for review April 30, 2012) Small cell lung cancer (SCLC) accounts for about 15% of all lung and “synthetic lethality” have emerged (15–19). As a complementary cancers. The prognosis of SCLC patients is devastating and no bi- approach, screening of libraries of small molecules across genomi- ologically targeted therapeutics are active in this tumor type. To cally characterized cell line panels has revealed direct oncogene develop a framework for development of specific SCLC-targeted dependencies as well as synthetic lethal dependencies (20–23). drugs we conducted a combined genomic and pharmacological vul- The use of small molecules offers the advantage of immediately nerability screen in SCLC cell lines. We show that SCLC cell lines addressing the question of whether a given vulnerability can be capture the genomic landscape of primary SCLC tumors and pro- chemically attacked. vide genetic predictors for activity of clinically relevant inhibitors To identify therapeutically relevant genome alterations in by screening 267 compounds across 44 of these cell lines. We show SCLC, we performed a combined genomic and chemical vul- MYC Aurora kinase inhibitors are effective in SCLC cell lines bearing nerability analysis in a panel of 60 SCLC cell lines. This study fi – MYC ampli cation, which occur in 3 7% of SCLC patients. In -ampli- involved the screening of a library of 267 compounds across 44 fi ed SCLC cells Aurora kinase inhibition associates with G2/M-ar- SCLC cell lines coupled to genomic characterization of these rest, inactivation of PI3-kinase (PI3K) signaling, and induction of and additional cell lines. apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of Results cytoplasmic levels of MYC. Our study suggests that a fraction of Similarity of SCLC Cell Lines and Primary Tumors. fi We analyzed SCLC patients may bene t from therapeutic inhibition of Aurora B. chromosomal gene copy number alterations in 60 patient-derived Thus, thorough chemical and genomic exploration of SCLC cell lines SCLC cell lines (Dataset S1) using Affymetrix 6.0 SNP arrays and may provide starting points for further development of rational determined significant copy number alterations using the pre- targeted therapeutic intervention in this deadly tumor type. viously described GISTIC algorithm (Dataset S2) (24, 25). Next, we compared the significant alterations present in the cell line ver the past years the development of targeted therapies has collection to the genetic alterations of a previously described – Odramatically affected clinical treatment of lung cancer (1 3). collection of 63 primary SCLC specimens (Fig. 1A) (13). Con- This development was sparked by the identification of mutations in fi EGFR – rming an overall high similarity of SCLC cell lines and primary (4 6) that confer exquisite sensitivity to EGFR inhibitors (2, tumors, this analysis revealed a significant (r = 0.83) correlation 7) and EML4-ALK fusions (8) that make tumors susceptible to ALK inhibition (3). The recent identification of FGFR1 amplification and DDR2 mutations in squamous cell lung cancer (SQLC) patients has Author contributions: M.L.S., F.D., M.P., and R.K.T. designed research; M.L.S., F.D., J.S., fueled hopes that not only lung tumors of never-smokers bear H.B.-W., C.M., M.K., S.H., F.M., J.M.H., P.M.S., and L.C.H. performed research; A.R., P.A.E., therapeutically amenable genetic alterations (9, 10). However, in R.T.U., A.P.A., V.V.V., T.N., H.W., and D.R. contributed new reagents/analytic tools; M.L.S., small cell lung cancer (SCLC) the lack of specimens suitable for F.D., M.P., J.S., H.B.-W., C.M., M.K., A.R., S.H., F.M., J.M.H., D.S., P.A.E., R.T.U., A.P.A., V.V.V., P.M.S., T.N., H.W., R.B., D.R., L.C.H., and R.K.T. analyzed data; and M.L.S., F.D., and R.K.T. deep genomic characterization has so far hampered similar efforts wrote the paper. to identify novel therapeutically relevant genome alterations. Conflict of interest statement: R.K.T. received consulting and lecture fees from Sanofi- Among the genes recurrently affected by genomic alterations in Aventis, Merck KGaA, Bayer, Lilly, Roche, Boehringer Ingelheim, Johnson & Johnson, SCLC are TP53, RB1,aswellastheMYCfamilygenessuchasMYC, AstraZeneca, Atlas-Biolabs, Daiichi-Sankyo, and Blackfield as well as research support MYCL1,andMYCN, which are frequently amplified in a mutually from AstraZeneca, Merck, and EOS. R.K.T. is a founder and shareholder of Blackfield, a company involved in cancer genome services and cancer genomics-based exclusive manner (11, 12). The PI3-kinase (PI3K) pathway has been drug discovery. proposed to be a therapeutically actionable signaling cascade that is This article is a PNAS Direct Submission. activated in SCLC (11) but the frequency of genetic alterations driving PI3-kinase activation is currently unclear (13). Furthermore, Freely available online through the PNAS open access option. the Hedgehog (HH) pathway has been identified as a potentially Data deposition: The SNP array data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. druggable target in SCLC mouse models (14) but it is presently GSE40142). unclear whether HH signaling dependency segregates with partic- 1M.L.S. and F.D. contributed equally to this work. ular genetic alterations. 2To whom correspondence may be addressed. E-mail: [email protected] or roman. Given the inherent difficulties in the rational design of potent [email protected]. inhibitors of MYC and other transcription factors, alternative ther- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. apeutic strategies such as inhibition of MYC-MAX dimerization 1073/pnas.1207310109/-/DCSupplemental. 17034–17039 | PNAS | October 16, 2012 | vol. 109 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1207310109 Downloaded by guest on September 24, 2021 associated with the stage of the tumor as seen previously for MYC (Fig. 1 A–C and Dataset S3) (28, 29). However, cell line artifacts and a treatment bias might contribute to this association and cannot be formally excluded. To confirm our findings of significant copy number changes in SCLC, we analyzed an independent cohort of 55 primary SCLC tissues for the presence of MYC amplification using FISH (Fig. 1D). In accordance with published data (25), we identified high-level amplification of the MYC gene in about 5.5% of primary SCLC samples (Fig. 1 D and E). Thus, our data suggest that our cell line collection captures major copy number alterations of SCLC. Activity Profiles of Clinically Relevant Targeted Compounds Across SCLC Cell Lines. We performed a systematic cell-based screen (44 SCLC cell lines) against a library of 267 compounds with diverse scaffolds (Fig. 2A), targeting a wide range of cellular proteins (Dataset S4 and SI Appendix,Fig.S2A) (30–34). Compound ac- tivity was assessed across cell lines as the remaining cellular via- bility at two different concentrations (Dataset S5). The resulting activity profiles ranged from compounds with no activity (n = 97) at high concentrations (5–10 μM) across all cell lines to com- pounds with high activity at low concentrations (0.5–1 μM) across the majority of cells (e.g., IPI-504) to highly selective compounds (e.g., PD173074 and PD0325904) (SI Appendix,Fig.S2B and Dataset S5) showing activity in only a few cell lines. Using hierarchical clustering of the raw inhibitor activity data, we identified compound groups of different scaffolds indicating common targets (Fig. 2B). For example, the mTOR inhibitor everolimus shared a cluster with the AKT inhibitor MK-2206, the PI3K inhibitor PI-103, and the spirooxindole derivative AA123, previously described to induce mitotic arrest in cellular assays (Fig. 2B and Dataset S4) (30). Our data therefore suggest that AA123 might be a scaffold that inhibits the PI3K-signaling path- way. This analysis supports the robustness of our screening ap- fi fl proach and affords identi cation of unexpected cellular targets Fig. 1. SCLC cell line collection re ects major genetic lesions of SCLC for unique compounds.
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