A Framework for Identification of Actionable Cancer Genome

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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 activity was assessed across cell lines as the remaining cellular viability 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 compounds 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 pathway. 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. patients. (A) Significant copy number changes (amplifications are in the fi To identify genetic predictors for the activity of the screened lower panel and deletions are in the upper panel) as de ned by GISTIC (q – values) in SCLC primary samples (green and brown) and in SCLC cells (red and compounds, we used signal-to-noise based feature selection blue). Selected genes are annotated. (B) Significant amplifications (exten- combined with the K-nearest-neighbor (KNN) algorithm (22) sive, red; limited, black) as defined by the GISTIC algorithm (g score) iden- (Dataset S6). This analysis revealed that PTEN loss predicts cy- tified in SCLC tumors. (C) Copy number changes at the MYC locus are totoxic activity of the HSP90 inhibitor IPI-504 and its close ho- displayed for the top 10 MYC amplified of extensive- (Left) and limited molog 17-AAG (P = 0.02 and P = 0.01; Fisher’s exact) (Dataset (Right)-stage samples. (D) FISH analysis of a sample showing amplification of S6). Surprisingly, PTEN loss did not predict efficacy of PI3K fi MYC (Left) and a sample with no MYC ampli cation (Right) (MYC, green; inhibitors (Dataset S6). Overall, these results suggest that in the control, red). (E) Frequency of MYC amplification in primary samples as PTEN fi determined by SNP arrays in a published dataset (25) and by FISH in the clinical setting loss may be a genetic marker for the ef cacy independent SCLC cohort. of HSP90 inhibitors but not PI3K inhibitors in SCLC. Next, we identified FGFR1 amplification as a predictor for the activity (P = 0.05) of the FGFR inhibitor PD173074 (Dataset S6). of copy number alterations in both datasets (Fig. 1A and SI Ap- FGFR1 amplification is a recurrent genome alteration in SQLC, pendix,Fig.S1A), similar to findings in other tumor types (20–22). associated with FGFR dependency in some lung cancer cell lines Our cell line collection captures hallmark events of SCLC such as (9, 11, 35). To test whether FGFR1 amplification is also linked with recurrent deletions of RB1 and PTEN (13, 26) but also amplifica- cytotoxic activity of FGFR inhibition in SCLC, we determined the C tion of genes such as FGFR1 (11, 13). Furthermore, in both GI50 values for a subset of seven SCLC cells (Fig. 2 ). One of the FGFR1 fi datasets we identified recurrent and focal amplification of MYCL1, two -ampli ed cell lines, DMS114, was previously shown to = µ C MYCN MYC MYCN fi be sensitive to PD173074 (GI50 0.46 M) (9) (Fig. 2 ). By ,and (13). High-level ampli cation (inferred FGFR1 fi copy number > 4) occurred in about 4–6% of cases in both data- contrast, the other -ampli ed cell line, SBC7, was resistant sets, whereas MYCL1 (primary samples, 8% and cell lines, 22%) to PD173074 and no apoptosis was induced upon treatment with the FGFR inhibitor (Fig. 2 C and D and SI Appendix,Fig.S3A). (Dataset S3)andMYC amplification (primary samples, 3% and These data suggest that not all FGFR1-amplified SCLC tumors cell lines, 15%) was detected with a higher prevalence in SCLC cell may be dependent on FGFR1 activity. Because these cells lack lines (Fig. 1 A–C and Dataset S3) (27). Although major events such SI Appendix B MYC fi expression of PTEN ( ,Fig.S3 ), we speculate that as ampli cation are found in both datasets, overall the sig- PTEN loss may contribute to modulation of FGFR inhibitor sen- fi ni cant copy number changes of SCLC differ from those found in sitivity. Consequently, despite inducing dephosphorylation of the r = SI Appendix non-small cell lung cancer (NSCLC) ( 0.57) ( ,Fig. adaptor protein FRS2 in SBC7 cells (Fig. 2E), PD173074 induced B C S1 and ). Given the high prevalence of limited stage disease apoptosis in DMS114 cells but not in SBC7 cells (Fig. 2D). (∼68%) in the cohort of patient samples and the high prevalence of advanced stage disease in the case of the cell lines (∼95%) the MYC Amplification Predicts Efficacy of Aurora Kinase Inhibitors. Us- frequency of MYCL1 (Dataset S3)andMYC amplification is likely ing the KNN algorithm, we next sought to identify compounds MEDICAL SCIENCES

Sos et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 17035 Downloaded by guest on September 24, 2021 Fig. 2. Identiﬁcation of therapeutically tractable alterations in SCLC. (A) All screened chemical scaffold groups are depicted. (B) Hierarchical clustering of the raw activity data across all SCLC cell lines and compounds (0.5–1 μM) showing activity (red, high; white, low) in at least one cell line (viability <50%; 0.5–1 μM).

Selected genetic lesions (Lower). (black, present; gray, not present). (C)GI50 values for PD173074 after 96-h treatment in SCLC cells are displayed (red, FGFR1 amplified; black, FGFR1 nonamplified). (D) Induction of apoptosis after 72-h treatment with 1 μM of PD173074 as assessed by FACS (annexin V/PI) is displayed for two FGFR1-amplified cell lines. (E) SBC7 cells treated for 24 h with PD173074 were analyzed for protein expression of phospho-FGFR, FGFR, phospho-FRS2, PARP, and actin by immunoblotting.

with specific activity in MYC-amplified cell lines. We found the (P = 0.004 MLN8237; P = 0.003 PHA680632; P = 0.01 VX680; PLK1 inhibitor BI2536, the ROCK1 inhibitor GSK269962A, as P = 0.01 ZM447439) enrichment of MYC-amplified cells in the well as the pan-Aurora inhibitor VX680 to be specifically active subgroup of sensitive cells (<1 μM) for all four Aurora kinase in these cells (Datasets S5, S6, and S7) (36). To test whether inhibitors (Fig. 3A). We next assessed the ability of VX680 (37) to Aurora kinase inhibitor efficacy is linked to MYC amplification in induce apoptosis in a subset of 11 cell lines. Using flow cytometry, SCLC, we determined the GI50 values of structurally diverse we observed robust induction of apoptosis after 24–48 h of treat- Aurora kinase inhibitors VX680, MLN8237, PHA680632, and ment with VX680 in MYC-amplified cell lines but not in those ZM447439 (Fig. 3A and Dataset S7). We observed a significant lacking MYC amplification (Fig. 3B). Furthermore, Aurora

17036 | www.pnas.org/cgi/doi/10.1073/pnas.1207310109 Sos et al. Downloaded by guest on September 24, 2021 Fig. 3. Inhibition of Aurora kinases leads to cell

death and apoptosis in MYC-amplified cells. (A)GI50 values (Dataset S7) for the Aurora kinase inhibitors MLN8237, PHA680632, VX680, and ZM447439 (96-h treatment) in a subset of 34 SCLC cell lines (*MYC amplified). P values (Fisher’s exact test) are displayed. (B) Induction of apoptosis after treatment with 1 μM of VX680 (12–48 h) as assessed by FACS (annexin V/PI) is displayed for six MYC-amplified and five MYC-nonamplified cell lines. Inset shows representative pictures of GLC1 and SBC6 cells treated with either control or VX680. (C) Depicted is the fraction of cells in the G2/M-phase as measured by flow cytometry (PI signal) in six MYC-amplified (black) and five MYC-nonamplified cell lines (white). Error bars represent SD. (D) GLC1, N417, and SBC6 cells treated with VX680 (24 h) were analyzed for protein expression of Aurora A, phospho-HH3, PARP, phospho-AKT, AKT, MYC, and actin by immunoblotting. Lysates for detection of phospho-AURKA/B/ Cweresonificated.

inhibition induced a collapse of the mitochondrial membrane po- 3D). The observed induction of apoptosis translated into inhibition tential, specifically in MYC-amplified SCLC cells (SI Appendix, of tumor growth of VX680-treated (60 mg/kg) mice engrafted with Fig. S4A). Inhibition of Aurora kinases also led to significantly MYC-amplified cells (GLC1) (SI Appendix,Fig.S5). By contrast, faster G2/M-arrest in MYC-amplified SCLC cell lines, compatible no growth inhibition was observed in nude mice engrafted with with a generally increased proliferation rate in these cells (Fig. 3C). MYC-nonamplified SW1271 cells (SI Appendix,Fig.S5). Of note, To test whether MYC-amplified SCLC cells are generally vulner- increasing concentrations of VX680 led to enhanced levels of p- able to induction of a G2/M-arrest, we measured apoptosis after AKT in the control cell line, indicating that in MYC-amplified cells treatment with the prometaphase-arresting agent nocodazole. No a lack of feedback loops that activate the PI3K pathway may difference between the induction of apoptosis with nocodazole or contribute to the Aurora dependency (Fig. 3D). Depletion of VX680 was observed in MYC-nonamplified SCLC cells, whereas in Aurora A was recently shown to destabilize MYCN protein in MYC-amplified cells, we measured a significant difference (Wil- MYCN-amplified neuroblastoma; the catalytical activity of Aurora coxon test; P = 0.025) between nocodazole and VX680 (SI Ap- A was, however, not required (15). By contrast, in MYC-amplified pendix,Fig.S4B), suggesting that VX680-induced cell cycle arrest SCLC, inhibition of Aurora kinase activity did not affect MYC is not the main driver of cytotoxicity in MYC-amplified cells. protein levels (Fig. 3D). Overall, these data indicate that Aurora VX680 treatment led to dephosphorylation of Aurora A/B/C as kinase activity is specifically required for the survival of MYC- well as of histone H3 (HH3), a surrogate marker of Aurora B amplified SCLC cells and that the mechanism differs from Aurora signaling, in all cell lines at concentrations in the range of the de- dependency in MYCN-amplified neuroblastoma. termined GI50 values (Fig. 3 A and D). Aurora inhibition was paralleled by poly(ADP-ribose)-polymerase (PARP) cleavage and Dependency on AURKB Activity in MYC-Amplified SCLC Cells. MYC is a reduction of phosphorylation of AKT in MYC-amplified cells known to be involved in the pathogenesis of diverse cancer types (GLC1 and N417) but not in MYC-nonamplified SBC6 cells (Fig. (38). To test the relevance of MYC amplification in SCLC, we MEDICAL SCIENCES

Sos et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 17037 Downloaded by guest on September 24, 2021 of AURKB but not of AURKA induced PARP cleavage in MYC- amplified SCLC cell lines (Fig. 4C). Note that the AURKA-targeted hairpin 3 also has off-target effects against AURKB (Fig. 4C, Left). The observed dependency on AURKB expression was cor- related with MYC dependency (r = 0.96) (SI Appendix,Fig.S6E). As a consequence, knockdown of AURKB (Fig. 4D) but not AURKA (Fig. 4E) resulted in a reduction of cell viability in MYC- amplified SCLC cells. As a further confirmation of preferential dependency of MYC-amplified SCLC cells on Aurora B, we next tested barasertib-hQPA (AZD1152), a compound with ∼1,000- fold selectivity against Aurora B compared with Aurora A (39). AZD1152 induced marked dephosphorylation of Aurora B and partially Aurora C but not Aurora A at concentrations of 0.1 μMin all cell lines (Fig. 4F). However, AZD1152 treatment led to a reduction of cell viability at low nanomolar concentrations only in the two MYC-amplified but not in MYC-nonamplified cells (Fig. 4G). Thus, further extending results obtained in genetically engineered cells (36), the observed activity of Aurora kinase inhibition is predominantly mediated by inhibition of Aurora B in MYC- dependent cells. Discussion The findings of this combined genomic and chemical vulnerability screen support the use of this approach to develop strategies for genetically tailored therapies against SCLC. We demonstrate that our cell line panel captures the major copy number alterations of primary SCLC tumors, thereby allowing extrapolating to actual patient populations. Furthermore, the diversity of the screened inhibitors provides a first broad assessment of pathway dependencies across representative SCLC genotypes. Building on previous studies (11, 40), the scaling of both the number of cell lines and the number of compounds afforded identification of vulnerabilities associated with infrequent genome alterations, such as amplifications of FGFR1 MYC FGFR1 fi Fig. 4. MYC-amplified SCLC cells depend on MYC and Aurora B protein and . In the case of ampli cation, further studies will be expression. (A) Viability of SCLC cells after transduction with MYC-shRNA required to clarify the frequency of FGFR1 dependency in SCLC, selected from a set of four shRNA constructs (SI Appendix, Fig. S6A)com- as other genetic lesions may play a role in the responsiveness to pared with controls is displayed. Error bars represent SD. (B)GLC1,H211, FGFR inhibition. N417, and SBC6 cells after transduction with either control or MYC-shRNA Furthermore, we describe and functionally characterize the were analyzed for MYC and PARP protein by immunoblotting. (C) N417 dependency of MYC-amplified SCLC tumors on Aurora B (41). (Left) and SW1271 (Right) cells after transduction with either control or We find that MYC-amplified tumors depend on the kinase activity AURKA/B-shRNA constructs were analyzed for protein expression of Aurora of Aurora B for their survival (36). Currently a series of Aurora A/B, PARP, and MYC by immunoblotting. (D) Viability of SCLC cells after kinase inhibitors (e.g., MLN8237 and PHA739358) including the transduction with AURKB-shRNA or AURKA-shRNA (E) compared with controls was assessed. (F) N417, GLC1, SW1271, and SBC4 cells treated with Aurora B-selective inhibitor barasertib (AZD1152) are un- AZD1152 (24 h) were analyzed for protein expression of phospho-Aurora A/ dergoing clinical evaluation in phase I/II studies (42, 43). Our B/C, Aurora A/B, and actin by immunoblotting. Lysates for detection of data provide a rationale for the testing of these compounds in phospho-AURKA/B/C were sonificated. (G) Viability of N417, GLC1, (MYC- genetically defined SCLC patient groups. amp, red) SW1271, and SBC4 (MYC-nonamp, black) cells after 96-h treat- Previous studies did not support a role for Aurora dependency in ment with barasertib (AZD1152) was determined with celltiter-glo (CTG) NSCLC cell lines of different genotpyes (22) or implied different assays. Error bars represent SD. MYC family genes in a mixed lung cancer panel (40), thus indicating that the role of amplified MYC and its dependency on AURKB (36) may differ between different cancer subtypes. Thus, our data pro- silenced expression of MYC (SI Appendix,Fig.S6A) in a subset of fi A vide a lineage-speci c extension of previous data generated in SCLC cell lines and assessed their viability (Fig. 4 ). We ob- lymphoma mouse models driven by an active transgene of MYC MYC fi served a strong association between ampli cation and MYC (36). Compatible with this notion is the recent finding that BRAF MYC fi dependency resulting in induction of cell death in -ampli ed mutations associate with BRAF inhibitor sensitivity in melanoma MYC- fi A but not in nonampli ed cells (Fig. 4 ). The MYC de- but not colorectal cancer (44, 45). Our data not only point out the MYC fi SI Appendix B pendency of -ampli ed cells ( ,Fig.S6 ) was also differences in the biology of distinct subtypes of lung cancer but also linked to gene expression of MYC but not AURKA and AURKB underscore the differences in oncogenic signaling of MYC gene (SI Appendix,Fig.S6C) and independent of MYC, Aurora A, and family members: although amplifications of MYCL1 and MYCN Aurora B protein expression (SI Appendix,Fig.S6D). Knockdown amplifications occur in a mutually exclusive fashion with MYC of MYC led to induction of apoptosis in MYC-amplified cells as amplification (suggesting genetic epistasis), they do not segregate assessed by PARP cleavage in immunoblotting assays (Fig. 4B). with vulnerability to Aurora B inhibition in SCLC. To validate our findings, we silenced expression of AURKA and Although MYC-amplified SCLC represents only a small subset AURKB in a panel of SCLC cell lines (Fig. 4C). We had observed of lung cancer patients, recent experience with the successful in- preferential but subtle dephosphorylation of Aurora B over Au- troduction of ALK inhibitors for the treatment of about 2–3% rora A in two of the cell lines treated with VX680 (Fig. 3D, Center ALK fusion positive adenocarcinomas suggest that genetic strati- and Left), compatible with requirement of Aurora B in the context fication is feasible and beneficial, even in small subgroups (3). Our of MYC amplification in SCLC. Supporting this notion, knockdown study provides a framework for preclinical testing of genetically

17038 | www.pnas.org/cgi/doi/10.1073/pnas.1207310109 Sos et al. Downloaded by guest on September 24, 2021 encrypted vulnerabilities in SCLC. In support of this notion, our triplicates and compared with DMSO controls. Calculation of the P values was initial screen has already yielded actionable targets for further performed using a Wilcoxon rank sum test, a two-tailed t test, or a Fisher’s preclinical validation and possible clinical testing. We therefore exact test implemented in “R”. Pharmacodynamic response of signaling was hope that—in the longer term—our approach might help to im- measured by immunoblotting of cellular lysates of treated cells using phospho- prove the disappointing survival rates of these patients. speciﬁc antibodies. Not all bands were detected at the same membrane due to overlapping protein sizes. RT-PCR assays were performed with SYBR Green and Methods primers for the respective genes and GAPDH as control. For gene silencing, – All patients gave written informed consent sample analysis. The tumor speci- lentiviruses were produced with pLKO.1-Puro based vectors. After trans- mens have been collected under local institutional review board approval duction, cell viability was measured by measuring cell numbers of quad- (University of Cologne, Cologne, Germany) and genomic analyses were per- ruplicates and normalized to viability of cell lines transduced with control formed as described elsewhere (13). GISTIC analyses of a group of 60 SCLC cell constructs. All animal procedures were performed in agreement with the an- lines were performed as described previously (24, 25). For a subgroup of 36 cell imal protection committee and the local authorities and treatment was per- lines, previously published copy number data (www.sanger.ac.uk/genetics/ formed as described previously (22). CGP/CellLines) were used. The human genome build hg18 was used. All raw copy number data have been deposited in the Gene Expression Omnibus da- ACKNOWLEDGMENTS. We thank Drs. Christian Reinhardt and Hamid tabase (accession no. GSE40142). The cell line collection of 44 unique small cell Kashkar for support and AstraZeneca for providing barasertib-hQPA lung cancer patient-derived cells was used for cell-based screening against 267 (AZD1152). This work was supported by the European Union-Framework inhibitors using CellTiterGlo as a growth inhibition assay. Detailed analysis was Programme CURELUNG (HEALTH-F2-2010-258677 to R.K.T.); by the Deut- performed on a subset of 51 compounds that showed activity in at least one cell sche Forschungsgemeinschaft through TH1386/3-1 (to R.K.T. and M.L.S.) and through SFB832 (TP6 to R.K.T. and TP5 to L.C.H.); by the German Min- line at concentrations of 0.5–1 μM. Annexin V and propidium iodide (PI) ﬂ istry of Science and Education as part of the National Network for the Study staining was used to measure apoptosis in ow cytometry assays. For the of the Genome (NGFN) plus program (Grant 01GS08100 to R.K.T.), by the subgroup of cell lines where the raw copy number data were not generated in Max Planck Society and by the Behrensweise Foundation (M.I.F.A. house, genotyping of a panel of 11 SNPs was performed to control the re- NEUR8061 to R.K.T.); and by an anonymous foundation (R.K.T.). M.L.S. is spective annotation. Cell viability was measured at two concentrations in a fellow of the International Association for the Study of Lung Cancer.

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