Genetic Modifiers of EGFR Dependence in Non-Small Cell Lung Cancer

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Genetic modifiers of EGFR dependence in non-small cell lung cancer

Tanaz Sharifniaa,b,c, Victor Rusua,d, Federica Piccionia, Mukta Bagula, Marcin Imielinskia,b,c, Andrew D. Cherniacka, Chandra Sekhar Pedamallua,c, Bang Wonga, Frederick H. Wilsona,c, Levi A. Garrawaya,c, David Altshulera,d,e, Todd R. Goluba, David E. Roota, Aravind Subramaniana, and Matthew Meyersona,b,c,1

aBroad Institute of Harvard and MIT, Cambridge, MA 02142; Departments of bPathology and dGenetics and Medicine, Harvard Medical School, Boston, MA 02115; cDepartment of Medical Oncology, Dana–Farber Cancer Institute, Boston, MA 02115; and eDepartment of Molecular Biology and Diabetes Unit, Massachusetts General Hospital, Boston, MA 02114

Edited by Bert Vogelstein, Johns Hopkins University, Baltimore, MD, and approved November 18, 2014 (received for review June 29, 2014)

Lung adenocarcinomas harboring activating mutations in the achieved via EGFR activation in MET-dependent cells (13), and epidermal growth factor receptor (EGFR) represent a common mo- analogous examples of reciprocal kinase switching have been lecular subset of non-small cell lung cancer (NSCLC) cases. EGFR reported in other kinase-driven cancer models (14, 15). These and mutations predict sensitivity to EGFR tyrosine kinase inhibitors other findings suggest that compensatory kinase switching may be (TKIs) and thus represent a dependency in NSCLCs harboring these a more general way in which oncogene-dependent cancers over- alterations, but the genetic basis of EGFR dependence is not fully come reliance on their primary driver kinase (14, 16), but the full- understood. Here, we applied an unbiased, ORF-based screen to range of kinases capable of mediating EGFR bypass has not been identify genetic modifiers of EGFR dependence in EGFR-mutant systematically studied. Recent advances in large-scale functional genetic libraries have NSCLC cells. This approach identified 18 kinase and kinase-related made it possible to query a wide range of genetic perturbations for genes whose overexpression can substitute for EGFR in EGFR- their ability to modulate specific cellular phenotypes in mammalian dependent PC9 cells, and these genes include seven of nine Src EGFR FGFR1 FGFR2 ITK NTRK1 NTRK2 MOS systems (17, 18). Using the model of -mutant, erlotinib- family kinase genes, , , , , , , sensitive NSCLC cells, we have performed a systematic ORF-based MST1R RAF1 , and . A subset of these genes can complement loss screen to identify kinase and kinase-related genes whose over- of EGFR activity across multiple EGFR-dependent models. Unbiased expression can complement loss of EGFR activity in an EGFR- gene-expression profiling of cells overexpressing EGFR bypass dependent context. Our findings indicate broad potential for EGFR genes, together with targeted validation studies, reveals EGFR- substitution in the setting of EGFR dependence, with compensatory independent activation of the MEK-ERK and phosphoinositide mechanisms commonly conferring EGFR-independent activation of 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR the PI3K-AKT and MEK-ERK signaling pathways. Importantly, this and MEK restores EGFR dependence in cells expressing each of approach has recovered known mechanisms of erlotinib resistance as the 18 EGFR bypass genes. Together, these data uncover a broad well as identified novel mediators of EGFR bypass in EGFR-mutant spectrum of kinases capable of overcoming dependence on EGFR NSCLC. These data support the idea that the EGFR-dependent and underscore their convergence on the PI3K-AKT and MEK-ERK state can be redundantly driven by diverse genetic inputs that signaling axes in sustaining EGFR-independent survival. commonly converge on shared downstream signaling nodes. Results epidermal growth factor receptor | non-small cell lung cancer | ORF An ORF-Based Screen Identifies 18 Genes Whose Expression Can Substitute for EGFR in an EGFR-Dependent NSCLC Cell Line. To he term “oncogene addiction” has been used to describe the identify genes that complement loss of EGFR activity in the Tphenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions (1). In non-small Significance cell lung cancer (NSCLC), this principle is perhaps best exem- plified with the finding that epidermal growth factor receptor Non-small cell lung cancers (NSCLCs) harboring mutations in (EGFR) mutations predict response to EGFR tyrosine kinase the epidermal growth factor receptor (EGFR) gene are often inhibitors (TKIs) gefitinib and erlotinib, and thus represent a de- singularly reliant on EGFR activity for tumor cell survival, but pendency in the subset of tumors harboring these alterations (2–6). the genetic basis for this dependence is not fully understood. In However, though EGFR-mutant NSCLCs typically respond dra- this study, we have performed a screen to identify a spectrum matically to EGFR TKIs, clinical responses are not universal, even of kinase genes whose overexpression can overcome NSCLC within this genetically defined cohort, with the rate of objective cells’ reliance on EGFR. Using both unbiased and targeted response estimated to be ∼71% (5, 6). Furthermore, the over- approaches, we demonstrate that these genes commonly by- whelming majority of patients who initially respond to EGFR pass dependence on EGFR through reactivation of downstream inhibitors ultimately develop resistance to therapy (7). A deeper signaling pathways. understanding of the genetic underpinnings of EGFR addiction, and how EGFR-mutant cells can overcome reliance on EGFR, may Author contributions: T.S., A.S., and M.M. designed research; T.S. and M.B. performed research; V.R., B.W., F.H.W., L.A.G., D.A., T.R.G., D.E.R., and A.S. contributed new re- improve clinical outcomes. agents/analytic tools; T.S., V.R., F.P., M.B., M.I., A.D.C., and C.S.P. analyzed data; F.P. Here, we have applied an unbiased screening approach to iden- supervised screening; B.W. visualized data; F.H.W. and L.A.G. guided anaplastic lym- tify genetic modifiers of EGFR dependence in NSCLC. Mounting phoma kinase studies; and T.S. and M.M. wrote the paper. evidence supports the existence of several genetic modifiers of Conflict of interest statement: M.M. was a consultant and received research support from EGFR dependence in EGFR-mutant NSCLC, which can reduce Novartis, and receives research support from Bayer. M.M., L.A.G., and T.R.G. are equity the degree to which these tumors rely on EGFR and thereby holders in, and consultants for, Foundation Medicine. contribute to EGFR TKI resistance (8). Examples include ampli- This article is a PNAS Direct Submission. fication of the MET receptor tyrosine kinase (RTK) (9), activation 1To whom correspondence should be addressed. Email: matthew_meyerson@dfci.

of the NF-κB pathway (8), amplification of the HER2 (ERBB2) harvard.edu. GENETICS RTK (10), amplification of the CRKL gene (11), and activation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the AXL kinase (12). Notably, MET bypass can be reciprocally 1073/pnas.1412228112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1412228112 PNAS | December 30, 2014 | vol. 111 | no. 52 | 18661–18666 Downloaded by guest on September 23, 2021 setting of EGFR dependence, we performed an ORF over- were assayed for erlotinib sensitivity across multiple drug doses expression screen in an EGFR-dependent NSCLC cell line in the using 72-h growth inhibition assays (Fig. 2A). For comparison, presence of erlotinib. A library of 589 ORFs encoding human the erlotinib-resistant EGFR-mutant positive controls EGFR- kinases and kinase-related proteins [Center for Cancer Systems Δ(E746-A750)-T790M (henceforth EGFR-ex19del-T790M) and Biology (CCSB)/Broad Institute Kinase ORF Collection] (17, EGFR-L858R-T790M were tested in parallel. Ectopic expression 18) was expressed via lentiviral transduction in the EGFR- of 18 of the 19 candidate bypass ORFs was capable of reducing mutant NSCLC cell line, PC9, which is sensitive to EGFR TKIs erlotinib sensitivity relative to cells transduced with an inert ORF A > with an IC50 of ∼30 nM (19). ORF-expressing PC9 cells were (Fig. 2 ). This reduced sensitivity corresponded to a twofold to treated with 300 nM erlotinib, 3 μM erlotinib, or DMSO for 72 h >400-fold shift in IC50 values (Table S1). One primary screening before being assayed for cell viability. Experimental ORFs were hit, YES1, failed to bypass EGFR dependence in our validation screened alongside positive control EGFR ORFs encoding the studies, leaving seven of nine Src family kinase genes confirmed to T790M “gatekeeper” mutation, which can promote resistance to confer the complementation phenotype (Fig. 2A and Table S1); EGFR TKIs (20), in cis with a canonical EGFR activating mu- and notably, this gene scored nearest to the cutoff we used to tation [EGFR-Δ(E746-A750)-T790M and EGFR-L858R-T790M] select ORFs for validation (Fig. 1). We noted that the known (21). Additional positive controls included ORFs encoding acti- EGFR bypass gene MET did not score in our primary screen, vating alleles of several MAPK family members. likely due to failure of the MET expression vector to express MET Cell viability under screening conditions was very low; the me- protein (Fig. S1B). Overall, we estimate that the vast majority of dian relative viability for all experimental ORFs was 12% at the screened ORFs express protein in PC9 cells (Fig. S1 C and D). 300-nM dose and 8% at the 3-μM dose (Fig. 1). Overexpression of The 18 genes capable of bypassing EGFR dependence in PC9 19 ORFs, of the 589 tested, led to a significant increase in viability cells were used to transduce four additional EGFR-mutant of erlotinib-treated PC9 cells, with viability of at least 39% in 300 NSCLC cell line models: the EGFR TKI-sensitive models nM erlotinib and at least 31% in 3 μM erlotinib (Fig. 1). The genes HCC827, HCC2935, and H3255 (22), and the partially sensitive whose ectopic expression led to greater viability in the presence of model HCC2279 (22, 23). Transduced cells were treated with 3 erlotinib include known modifiers of EGFR dependence (AXL, μM erlotinib and assayed for cell viability after 72 h (Fig. S2 A–E ERBB2, CRKL) as well as genes and gene families that are newly and Fig. 2B), and the effects of the 18 ORFs across all five identified candidate complementation partners of EGFR in models tested were grouped by gene and cell line (Fig. 2B). In- EGFR-mutant lung cancer cells, including eight of the nine Src terestingly, two classes of EGFR modifiers emerged from this family kinase members, as well as FGFR1 and FGFR2; ITK; analysis: one set of genes (CRKL, SRC, RAF1, FRK, BLK, and MST1R; MOS; NTRK1 and NTRK2;andRAF1.The19genes HCK, in addition to the erlotinib-resistant EGFR-ex19del- whose expression led to apparent EGFR substitution in the primary T790M mutant) whose overexpression could universally or screen were selected for validation and functional characterization. nearly universally reduce EGFR dependence across the models cDNA vectors corresponding to the 19 candidate EGFR by- tested (Fig. 2B, red cluster), and a second set of genes whose pass genes were sequence-verified and confirmed to express effects varied across different cell line models (Fig. 2B, blue protein (Fig. S1A). PC9 cells overexpressing each of these ORFs cluster). Overexpression of variable-effect cluster members LCK, FGR, LYN, and MST1R, and ITK, for example, each leads to increased cell viability in the presence of erlotinib in three of the five models tested (Fig. 2B, blue cluster). It is possible that the Experimental ORFs KRASV12 differing effects of the genes in the variable-effect cluster on 90 Mutant EGFR controls MEKDD EGFR dependence can be attributed to the transcriptional Mutant MAPK pathway controls FGR heterogeneity of these cell lines, which fall in distinct clusters HRASV12 Negative controls HCK when comparing expression profiles among lung cancer cell lines 80 (Fig. S3), as well as to varying strengths of gene-specific effects. No virus controls BLK CRKL SRC Importantly, the clinically validated EGFR TKI resistance- 70 AXL ERBB2 NTRK1 mediating genes and each modify EGFR dependence in only a subset of the models tested, suggesting that the effects of MST1R FGFR2 60 clinically relevant modifiers of EGFR dependence can also vary LY N AXL RAF1 EGFR MOS across different -mutant models. Together, these data NTRK2 identify 18 modifiers of EGFR dependence in PC9 cells, of which 50 ITK FRK FGFR1 a subset can universally complement loss of EGFR activity across ERBB2 multiple EGFR-dependent contexts. 40 YES1 Interestingly, in investigating whether EGFR bypass ORFs are LCK specific to EGFR TKIs, we observed that overexpression of 30 several EGFR bypass genes, including AXL, CRKL, ERBB2, MST1R RAF1 Percentage of control, 3 μM erlotinib μM 3 control, of Percentage , , and several Src family kinases, can overcome sen-

20 sitivity to the anaplastic lymphoma kinase (ALK) inhibitor TAE684 in EML4-ALK-positive H3122 NSCLC cells (Fig. S4A). In contrast, none of the EGFR bypass genes caused appreciable changes in 10 cellular response to the chemotherapy drug cisplatin in PC9 cells (Fig. S4B). These findings suggest that the kinases identified in this 0 screen do not universally mediate drug resistance, but they may 0 20 40 60 80 100 have a more general role in modifying oncogene dependence Percentage of control, 300 nM erlotinib in NSCLC.

Fig. 1. An ORF-based screen identifies 18 kinase and kinase-related modi- Inhibition of Bypass Kinases Restores Erlotinib Sensitivity, and fiers of EGFR dependence. Screening results for PC9 cells transduced with Coinhibition Is Associated with Phospho-AKT Attenuation for a Subset a library of kinase ORFs, treated with 300 nM or 3 μM erlotinib or vehicle, then assayed for cell viability after 72 h using CellTiter-Glo. Experimental ORFs of EGFR Bypass Genes. To enable the most inclusive analysis of (green points) were screened alongside EGFR-ex19del-T790M and EGFR-L858R- EGFR bypass genes, all 18 EGFR modifiers identified in PC9 T790M positive controls (yellow points); activating alleles of the MAPK family cells were carried forward for further study. First, because the (orange points); inert gene controls (red points); and no virus controls (blue modifier genes were predominantly kinases, we sought to de- points). Data are expressed as percent viability relative to vehicle. Nineteen termine whether the observed bypass phenotypes were kinase- candidate modifiers of EGFR dependence were selected for validation. dependent, as well as to verify the specificity of ORF activity, by

18662 | www.pnas.org/cgi/doi/10.1073/pnas.1412228112 Sharifnia et al. Downloaded by guest on September 23, 2021 AB150 LACZ LUCIFERASE 100 EGFR L858R T790M 50 EGFR ex19del T790M AXL 0 Experimental ORF 150 T790M 100 CRKL Fig. 2. Validation of the primary screen. (A) PC9 SRC 50 RAF1 cells expressing candidate EGFR bypass ORFs or BLK CRKL ERBB2 0 FRK controls were treated with erlotinib and assayed 150 BLK HCK for cell viability after 72 h using CellTiter-Glo. Data 100 LCK FGR are expressed as percent viability relative to vehi- 50 LY N cle-treated cells and represent the mean ± SD of FGFR1 FGFR2 FGR 0 MST1R four replicates. Graphs with identical control curves 150 NTRK1 FGFR2 reflect experiments performed in parallel on the 100 AXL MOS same day. (B) Heat map summarizing effects of 50 NTRK2 bypass ORFs validated in A across five EGFR-mutant FRK HCK ITK ITK 0 cell line models. The 18 EGFR bypass genes vali- 150 FGFR1

Percentage of control ERBB2 dated in PC9 cells were used for validation studies 100 LACZ in HCC2279, HCC2935, H3255, and HCC827 cells 50 (Fig. S2). Grayscale values represent increasingly LCK LYN MOS PC9 0 significant levels of ORF-induced erlotinib resis- 150 HCC827 HCC2279 HCC2935 H3255 tance relative to LACZ controls (black, 0; white, +4). 100 Data were generated from cells treated with 1 μM 50 Increased viability under erlotinib treatment, μ MST1R NTRK1 NTRK2 relative to LACZ (PC9) or 3 M erlotinib (HCC2279, HCC2935, H3255, 0 HCC827) (A and Fig. S2). Dendrograms in the row 150 P < 0.0001 0.0001 < P < 0.001 and column margins represent groupings of ORFs 100 0.001 < P < 0.01 and cell lines, respectively. ORFs segregate accord- 50 0.01 < P < 0.05 RAF1 SRC YES1 ing to whether their EGFR bypass-promoting 0 P ≥ 0.05 or no increase -4 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 effects are universal or nearly universal (red cluster) Log μM erlotinib or variable (blue cluster) across the cell lines tested. T790M denotes EGFR-ex19del-T790M.

testing whether enzymatic inhibition of bypass-promoting kinases a distinct PI3K-AKT and/or MEK-ERK signaling profile. ORF- could restore EGFR dependence. PC9 cells transduced with ki- expressing cells were treated with erlotinib, the relevant kinase nase ORFs were treated with erlotinib alone, the relevant kinase inhibitor, or their combination for 6 h, and assayed for down- inhibitor (where available) alone, or their combination for 72 h, stream signaling activation (Fig. 3 B–H). LACZ-expressing cells then assayed for cell viability (Fig. 3A and Fig. S5 A–G). Inhibitors undergo the expected down-regulation of phospho-EGFR, -AKT, tested included dasatinib (targeting the Src family kinases); XL880 and -ERK1/2 in the presence of erlotinib, in contrast to EGFR- (targeting AXL and MST1R); NVP-BGJ398 (BGJ398; targeting ex19del-T790M-expressing cells (Fig. 3B). Cells transduced with FGFR1 and FGFR2); lestaurtinib (targeting NTRK1 and Src family kinases, however, maintain AKT phosphorylation in the NTRK2); lapatinib (targeting ERBB2); BMS-509744 (targeting presence of 100 nM erlotinib, and this effect is reversed only upon ITK); and AZ628 (targeting RAF1). The on-target activity of cotreatment with dasatinib. Selective attenuation of phospho- these inhibitors was confirmed for a select number of ORFs (Fig. AKT under combination treatment was similarly observed in cells H–M S5 ). transduced with ERBB2, ITK, NTRK1, NTRK2, and very modestly Cells expressing Src family kinase genes could indeed be with FGFR1, FGFR2, AXL,andMST1R (Fig. 3 C–G). resensitized to erlotinib when cotreated with dasatinib, whereas Selective down-regulation of phospho-AKT was not observed these cells were not similarly sensitive to either agent alone (Fig. RAF1 A A in -expressing cells cotreated with erlotinib/AZ628 (Fig. 3 and Fig. S5 ). In contrast, resensitization to combined 3H), although as mentioned above, this combination was not erlotinib/dasatinib treatment was not achieved in cells expressing G EGFR sufficient to fully sensitize these cells (Fig. S5 ). Combined erloti- -ex19del-T790M. Gene-specific rescue of erlotinib sensi- nib/AZ628 treatment also fails to abolish phospho-ERK activation tivity was similarly observed for AXL; MST1R; FGFR1 and H FGFR2 NTRK1 NTRK2 ERBB2 ITK RAF1 in these cells (Fig. 3 ). Indeed, AZ628 treatment paradoxically ; and ; ; ; and upon induces phospho-RAF1 activation in RAF1-expressing cells (Fig. cotreatment with their respective inhibitors (Fig. 3A and Fig. S5 S5M), consistent with previous findings using RAF inhibitors in B–G). Combination treatment was also associated with increased other RAFWT models (24). levels of apoptosis in cells expressing bypass genes, compared with treatment with either inhibitor alone (Fig. S6). Together, EGFR Bypass Genes Commonly Induce Similar Transcriptional Effects, these data suggest that the EGFR bypass phenotype induced by these ORFs is specific to the encoded gene and requires the Which Are Anticorrelated to Those Induced by MEK and PI3K kinase activity of the expressed protein. Inhibitors. Given that a subset of kinase genes can substitute for A few kinase inhibitors showed somewhat different patterns of EGFR in PC9 cells, we were interested in identifying signaling growth inhibition or rescue. Lestaurtinib alone has some activity pathways that were potentially common to overexpression of in LACZ- and EGFR-ex19del-T790M-transduced cells (Fig. 3A these genes. A hypothesis-driven analysis described above in- and Fig. S5D), although only NTRK1- and NTRK2-expressing dicated a role for activation of the AKT pathway for a subset of cells exhibit enhanced sensitivity under combination treatment. ORFs; however, an unbiased systematic approach might more We also observed that AZ628 treatment only modestly resensi- clearly address whether EGFR bypass genes act through common tizes RAF1-transduced cells to erlotinib (Fig. 3A and Fig. S5G), or divergent pathways. We reasoned that a high-dimensional consistent with the finding that RAF1 overexpression is itself transcriptional readout would provide an unbiased approach to a mechanism of resistance to RAF inhibition (18). address this question. We assembled reagents for the 18 EGFR Next, because EGFR TKI treatment in EGFR-mutant cells bypass genes (positive-phenotype genes) as well as 19 kinase genes

typically elicits down-regulation of the PI3K-AKT and MEK- that had failed to modify EGFR dependence in the primary screen GENETICS ERK signaling pathways (2), we asked if rescue of EGFR de- (negative-phenotype genes). PC9 cells were transduced with these pendence using combination treatment was associated with vectors and controls for 72 h, then subjected to 24-h erlotinib

Sharifnia et al. PNAS | December 30, 2014 | vol. 111 | no. 52 | 18663 Downloaded by guest on September 23, 2021 LACZ T790M NTRK1 NTRK2 B LACZ T790M BLK FGR E erlotinib (100 nM) erlotinib (100 nM) dasatinib (100 nM) lestaurtinib (100 nM) Phospho-EGFR Phospho-EGFR Total EGFR A Total EGFR Phospho-AKT Phospho-AKT Total AKT Total AKT Phospho-ERK 1/2 erlotinibkinaseerlotinib inhibitor + kinase Phospho-ERK 1/2 inhibitor Total ERK 1/2 LACZ Total ERK 1/2 Fig. 3. Kinase inhibition of bypass-promoting kina- T790M BLK FRK HCK LCK LYN SRC LACZ T790M ERBB2 ses restores erlotinib sensitivity, and coinhibition is FGR erlotinib (100 nM) F erlotinib (100 nM) FRK dasatinib (100 nM) lapatinib (100 nM) associated with phospho-AKT attenuation for a sub- HCK Phospho-EGFR

dasatinib Phospho-EGFR LCK Total EGFR set of ORFs. (A) Inhibitor sensitivity of PC9 cells LYN Total EGFR Phospho-AKT SRC Phospho-AKT expressing indicated EGFR bypass-associated kinases Total AKT LACZ Total AKT and controls following treatment with erlotinib, the T790M Phospho-ERK 1/2 AXL Total ERK 1/2 Phospho-ERK 1/2 XL880 relevant kinase inhibitor, or their combination for MST1R Total ERK 1/2 LACZ 72 h. Cell viability was assayed with CellTiter-Glo, and AXL MST1R ITK T790M C LACZ T790M G LACZ T790M FGFR1 erlotinib (100 nM) erlotinib (1 μM) the resultant dose–response curves (Fig. S5) were

BGJ398 FGFR2 XL880 (100 nM) BMS-509744 (1 μM) LACZ Phospho-EGFR Phospho-EGFR used to generate area under curve (AUC) values, T790M Total EGFR Total EGFR plotted. Darker/lighter squares represent smaller/ NTRK1 Phospho-AKT Phospho-AKT NTRK2 – lestaurtinib larger AUC values. (B H) Immunoblot analysis of PC9 Total AKT Total AKT LACZ T790M Phospho-ERK 1/2 Phospho-ERK 1/2 cells expressing bypass-associated kinases and con- ERBB2 lapatinib Total ERK 1/2 Total ERK 1/2 trols under combination drug treatment. Transduced LACZ T790M LACZ T790M FGFR1 FGFR2 LACZ T790M RAF1 cells were treated with indicated doses of erlotinib, BMS- ITK D H 509744 erlotinib (100 nM) erlotinib (1 μM) LACZ BGJ398 (100 nM) AZ628 (1 μM) the relevant kinase inhibitor, or a combination for 6 T790M Phospho-EGFR Phospho-EGFR

AZ628 RAF1 h. Cells were incubated with 0.5% serum media 18 h Total EGFR Total EGFR before and during drug/DMSO treatment. Kinase AUC (Global) Phospho-AKT Phospho-AKT Total AKT Total AKT inhibitors tested included (B) dasatinib, (C) XL880, Min Max Phospho-ERK 1/2 Phospho-ERK 1/2 (D) BGJ398, (E) lestaurtinib, (F) lapatinib, (G) BMS- Total ERK 1/2 Total ERK 1/2 509744, and (H) AZ628. T790M denotes EGFR- ex19del-T790M.

treatment. Gene expression profiles for each ORF were used to transcriptional states induced by our panel of query genes were compute signatures as described in SI Materials and Methods. opposed to those induced by PI3K or MEK inhibition, and Unsupervised hierarchical clustering of these signatures yielded suggested that either or both of these pathways could be reac- A two distinct clusters (Fig. 4 ). Intriguingly, profiles segregated tivated in ORF-expressing cells. largely in accordance with positive vs. negative phenotypes; one subcluster was comprised solely of 12 bypass-associated profiles EGFR Modifier Genes Commonly Reactivate AKT and/or ERK1/2 EGFR A and the two double-mutant controls (Fig. 4 , red). Given Signaling, and PI3K-mTOR and MEK Coinhibition Restores EGFR that this cluster contained almost all bypass-associated gene profiles and none of the negative-phenotype profiles, we chose to Dependence. Given that PI3K and MEK inhibitor treatment sig- focus our subsequent analyses on the profiles represented in this natures were anticorrelated with those of our positive-phenotype A positive-phenotype cluster. query genes (Fig. 5 ), we asked whether EGFR bypass genes To address whether the expression profiles of cells transduced reactivate the PI3K-AKT or MEK-ERK pathways. PC9 cells with bypass-associated ORFs could yield insights into their biological effects, we used a large expression-profiling resource created by the Library of Integrated Network-Based Cellular Signatures (LINCS) program (www.lincscloud.org). This catalog was generated from A diverse human cell lines treated with a large number of genetic and chemical perturbagens. Gene expression signatures from cells expressing each of the 12 genes in the positive-phenotype cluster (excluding controls) were independently used to query LINCS. CC CC CAMK1D LYN STK40 JAK1 FGFR2 FGFR1 NTRK2 NLK LUCIFERASE MAPKAPK3 TESK2 CKS1B MAGI1 CDK5R1 AXL MAPK13 ITGB1BP3 LCK LOC652799 PRKCB ERBB2 MST1R STYK1 RAF1 MOS PDPK1 CSNK2A1 TAOK3 EGFR PRKAR2A FER CRKL LACZ ITK SRC NTRK1 FRK HCK FGR We first sought to identify LINCS ORFs whose transcriptional EGFR BLK L858R T790M effects positively correlate with query ORFs, to potentially ad- ex19del T790M dress whether EGFR bypass genes act through shared or distinct pathways. When considering the top ∼3% of positively correlated ORFs (Fig. 4B), query ORF signatures commonly correlate B Query Signature with LINCS signatures generated using the same ORF or from BLK CRKL FGR FRK HCK ITK LCK MOS NTRK1 NTRK2 RAF1 SRC SRC WWTR1 FGR TNFRSF1A LYN RAF1 LYN RAF1 RAF1 RAF1 RAF1 LYN other EGFR bypass ORFs (Fig. 4B, magenta cells). Notably, RARG FADD LYN LYN SRC CARD9 SRC IGF2 SRC MAGEB6 DDIT3 WWTR1 EGFR RAF1 FOS CRK SRC TIRAP FGR IFNG PITPNA BCL10 FOS MECP2 RAF1 none of the cell lines profiled in the LINCS dataset is IFNG RELB GPR139 RAF1 BCL10 CRK FGR CCND1 LYN NFE2L2 GRPR FGR TIRAP ZNF238 SRC CD40 LPAR1 SUOX GPR139 NQO2 IGF2 ZNF238 TP53 GPBAR1 mutant, and most are not of a lung lineage. Together with the FGR LPAR1 RAF1 BCL10 GPR139 FOS IFNB1 EIF4A2 TP53 ZRANB2 GPBAR1 SRC hierarchical clustering, these data suggest that a major subset of ELK3 CRK MAGEB6 CRK MAGEB6 FAS RAF1 PSMB10 RBM6 GPBAR1 ITGAE NFE2L2 VPS28 RAF1 TIRAP IFNG LTBR CCND1 TIRAP MECP2 FADD NDUFA13 GPR119 GPR139 EGFR bypass genes induce similar transcriptional effects, which do MAGEB6 CCND1 RELB FGR CD40 GPBAR1 MAGEB6 IL10 IFNG MECP2 WWTR1 LPAR1 EGFR PRDM1 SNAI3 FADD RELB FGR GPR139 CTBP1 CHRAC1 ADORA2A IL6R AKT1 PRKACA not appear to be restricted to an -mutant cellular context. Postively-correlated ORFs CDO1 SRC TNFRSF10B TIRAP RAF1 PPP1CA NFE2L2 RASD1 PSMB10 FOXA3 ZNF669 CRK The LINCS database also contains over 34,000 gene expres- CRK FGR TRAF2 GABPB2 CRK BRAF LPAR1 GRPR SQRDL PLK1 SMAD7 GPR119 sion profiles from cells treated with 3,103 compounds. We queried LINCS for chemical perturbations whose transcriptional Fig. 4. EGFR bypass genes commonly induce similar transcriptional effects. (A) Unsupervised hierarchical clustering of PC9 cells overexpressing bypass- effects were anticorrelated with those of query genes, hypothe- mediating ORFs (+); kinase ORFs unable to confer EGFR bypass (−); and sizing that such perturbations could represent compound classes controls (C). Twelve bypass-mediating ORFs displaying membership in a sin- with potential to reverse ORF-mediated effects. Among the top A gle cluster (positive-phenotype cluster, red) were used to query LINCS. (B) 0.7% anticorrelated compounds (Fig. 5 , blue bars), 13 of 21 LINCS ORFs whose signatures most positively correlate with those of positive- (>61%) could be classified as either MEK or PI3K inhibitors phenotype cluster genes. Each of the 12 positive-phenotype cluster genes (Fig. 5A, Lower Left). Along with the third-most abundant target, was used to independently query LINCS. The top ∼3% positively correlated SRC, these were the only recurrent drug targets represented at ORFs are listed in descending order. Query ORFs commonly correlate most the top of this list (Fig. S7A). These findings indicated that the strongly with the same ORF and/or other EGFR bypass genes (magenta).

18664 | www.pnas.org/cgi/doi/10.1073/pnas.1412228112 Sharifnia et al. Downloaded by guest on September 23, 2021 A Negatively correlated Positively correlated B EGFR LACZ ex19del T790M AXL BLK CRKL

0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10

0 0 0 0 -1 -0.8 -0.6 -0.4 -0.2 00.20.4 0.6 0.8 1 erlotinib (μM) 0 Median normalized rank Phospho-EGFR Total EGFR Fig. 5. EGFR bypass genes commonly reactivate Phospho-AKT

-1 -0.965 Total AKT AKT and/or ERK1/2 signaling, and PI3K-mTOR and All Phospho-ERK 1/2 MEK coinhibition restores EGFR dependence. (A) Total ERK 1/2 MEK Recurrent target PI3K Barcode plot displaying LINCS compounds ranked classes ERBB2 FGFR1 FGFR2 FGR FRK SRC by the correlations of their signatures to those of erlotinib (μM)

0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10

0 0 0 0 0 Phospho-EGFR C LACZ EGFR ex19del T790M AXL BLK positive-phenotype cluster ORFs. The most posi- 100 Total EGFR Phospho-AKT tively/negatively correlated compounds approach 50 Total AKT ranks of 1 and −1, respectively. The 0.7% most Phospho-ERK 1/2 0 Total ERK 1/2 negatively correlated compounds (blue portion of CRKL ERBB2 FGFR1 FGFR2 100 HCK ITK LCK LYN MOS barcode plot) include several inhibitors targeting

0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10

0 0 0 0 50 erlotinib (μM) 0 MEK, PI3K, and SRC (Fig. S7A). Each bar represents Phospho-EGFR 0 Total EGFR a chemical reagent. A single reagent targeting both Phospho-AKT FGR FRK HCK ITK PI3K and SRC is denoted with an asterisk. (B)Im- 100 Total AKT Phospho-ERK 1/2 munoblot analysis of PC9 cells overexpressing the 50 Total ERK 1/2 indicated ORFs and treated with erlotinib for 6 h. 0 MST1R NTRK1 NTRK2 RAF1 SRC Cells were incubated with 0.5% serum media 18 h Percentage of control LCK LYN MOS MST1R 100

erlotinib (μM) 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0 0 0 0 0 Phospho-EGFR before and during drug/DMSO treatment. (C) Cell 50 Total EGFR Phospho-AKT viability of PC9 cells overexpressing the indicated 0 Total AKT ORFs following treatment with 100 nM erlotinib NTRK1 NTRK2 RAF1 SRC Phospho-ERK 1/2 100 μ Total ERK 1/2 (erl), 500 nM of BEZ235 (BEZ), 2.5 M of AZD6244 50 (AZD), or their combinations for 72 h. Cell viability

0 was assayed with CellTiter-Glo. Data are expressed rl e erl erl erl BEZl/BEZAZD BEZ AZD BEZ AZD BEZ AZD as percent viability relative to vehicle-treated cells er erl/AZD erl/BEZ erl/AZD erl/BEZ erl/AZD erl/BEZ erl/AZD BEZ/AZD BEZ/AZD BEZ/AZD BEZ/AZD l/BEZ/AZD l/BEZ/AZD l/BEZ/AZD rl/BEZ/AZD er er er e and represent the mean ± SD of four replicates.

overexpressing EGFR bypass genes were treated with erlotinib expressing cell lines, with sensitivity often enhanced relative to for 6 h and profiled for activation of EGFR, AKT, and ERK1/2 erlotinib treatment in LACZ-expressing cells. In cases where at least (Fig. 5B). Indeed, persistent activation of AKT and/or ERK1/2 partial restoration of erlotinib sensitivity could be achieved using signaling under erlotinib treatment is a common feature of the either BEZ235 or AZD6244, resensitization was always potentiated majority of EGFR bypass genes, with sustained phospho-AKT by combining these two agents. Together, these data are consistent most prominently associated with BLK, FGR, FRK, HCK, LCK, with the concept that reactivation of AKT and ERK1/2 signaling SRC, ITK, and CRKL; and sustained phospho-ERK1/2 most commonly underlies ORF-mediated EGFR bypass. prominently associated with RAF1, BLK, ITK, MOS, AXL, and CRKL. Other ORFs, such as those encoding FGFR family genes, Discussion appear to display very modest activation of one or both of these We report the use of a systematic ORF-based screening ap- pathways, which may also be reflective of their more modest proach to identify the spectrum of kinases capable of bypassing rescue phenotype (Fig. 2A). We also observed that cells ex- EGFR dependence in EGFR-mutant NSCLC cells. To our pressing several kinases (BLK, LCK, FGR, and FRK) maintain knowledge, this represents the first cDNA-based genetic comple- EGFR phosphorylation in the presence of erlotinib; we found mentation screen for loss of EGFR in an EGFR-mutant model. that overexpression of these and other Src family kinases in PC9 This approach has identified known as well as previously un- cells can also modify a specific tyrosine residue, Y845, on EGFR identified drivers of EGFR bypass in EGFR-mutant NSCLC. More that is known to be the major site for c-Src-mediated phos- generally, it has revealed the breadth of kinases with compensatory phorylation (Fig. S8A) (25). potential in the setting of EGFR oncogene addiction: 18 genes for We similarly tested a representative subset of EGFR bypass PC9 cells, including both tyrosine and serine/threonine kinases. ORFs in H3255 cells for their ability to reactivate downstream Among the newly identified drivers of EGFR bypass in EGFR- pathways (Fig. S8B). Consistent with our findings in PC9 cells, mutant NSCLC, Src family kinases were highly represented, we observed varying degrees of phospho-ERK1/2 activation comprising over one-third of screening hits. The finding that Src under erlotinib treatment among kinases that can bypass EGFR family kinases are sufficient to modify EGFR dependence is in in this cell line. In contrast, we noted that several of the ORFs line with considerable evidence linking EGFR activity with this that fail to promote erlotinib resistance in H3255 cells do not family: c-Src itself has been characterized extensively with re- reactivate ERK1/2, although it was impossible to exclude the spect to its cooperative relationship with EGFR (27), and the possibility of ERK1/2 reactivation by LYN and NTRK2. The introduction of dominant-active c-Src can reduce the inhibitory observed differences in phospho-ERK1/2 activation in this cell effects of erlotinib in head and neck squamous cell carcinoma line potentially explain the sometimes varied resistance-inducing models (28). Src family kinase activation has been observed in effects of ORFs in different EGFR-mutant models. cetuximab-resistant colorectal adenocarcinoma and NSCLC To determine whether reactivation of these signal transducers squamous cell carcinoma in vitro models (29, 30), and it has been is required for ORF-induced bypass, PC9 cells transduced with reported that CRIPTO1-mediated EGFR TKI resistance in EGFR bypass genes were treated with erlotinib; the dual PI3K- NSCLC is Src-dependent (31). Furthermore, a recent report mTOR inhibitor NVP-BEZ235 (BEZ235); the MEK inhibitor described increased expression and activation of Src, mediated AZD6244; or their combinations, and assayed for cell viability by integrin activation, in EGFR TKI-resistant lung adenocarci- after 72 h (Fig. 5C). Drug doses of BEZ235 (500 nM) and noma models (32). The present study extends these findings to AZD6244 (2.5 μM) were determined empirically to be the lowest demonstrate that overexpression of SRC is itself sufficient to doses capable of maximally inhibiting AKT and ERK1/2 phos- bypass EGFR dependence in EGFR-dependent NSCLC, and phorylation (Fig. S7 B and C), inhibit the activity of their targets that this function is shared by seven of the nine family members. alone and in each of the combinations tested (Fig. S7D), and are Similarly, we noted that fibroblast growth factor receptor EGFR FGFR1 FGFR2 in similar ranges to those used by other investigators in - (FGFR) family kinase genes and , though not GENETICS mutant cell lines (26). Combined BEZ235/AZD6244 treatment previously recognized as sufficient for EGFR bypass, nonetheless was sufficient to restore sensitivity to erlotinib across all ORF- align with previous work describing up-regulation of FGFR1 as

Sharifnia et al. PNAS | December 30, 2014 | vol. 111 | no. 52 | 18665 Downloaded by guest on September 23, 2021 well as the ligand FGF2 in gefitinib-resistant NSCLC models, with (14, 15), with the finding that coactivation of multiple RTKs in concomitant dependency on the FGFR pathway (33, 34), and with glioblastoma cells overcomes reliance on any one RTK for down- other studies implicating FGF ligands in TKI resistance (15, 35). stream signaling activation (16), and with the identification of nine Other genes identified, including the neurotrophic tyrosine kinase-related genes whose overexpression can overcome RAF in- kinase receptor (NTKR) family kinases NTRK1 and NTRK2, the hibition in BRAF-mutant melanoma cells (18). Taken together, our Tec family kinase ITK, MST1R, and the serine/threonine kinases finding that a diverse set of kinases can redundantly drive the MOS and RAF1, have not previously been appreciated to modify EGFR-dependent state may thus represent a more general feature EGFR dependence in EGFR-mutant lung cancer cells, and thus of signal transduction in oncogene-dependent cancers. underscore the power of this screening approach in identifying novel mediators of bypass for a given dependency. Materials and Methods Identifying the spectrum of kinases capable of EGFR bypass Kinase ORF Screen. Screening was performed using a kinase ORF library of 589 is of considerable clinical interest given that patients with ORFs (CCSB/Broad Institute Kinase ORF Collection) (17, 18), along with the con- EGFR -mutant NSCLCs almost invariably acquire resistance to trols displayed in Fig. 1. Transduced PC9 cells were treated with 3 μM erlotinib, EGFR TKIs (7); a large fraction (30%) of acquired resistance 300 nM erlotinib, or DMSO, and cell viability was assayed 3 d later using CellTiter- cases are driven by unknown mechanism(s) (36); and because Glo (Promega). Additional details are described in SI Materials and Methods. mounting evidence suggests that activation of alternative driver kinases, such as MET, represents a common route by which Additional Materials and Methods. Additional materials and methods, in- kinase-driven cancers acquire resistance to therapy (9, 12, 18). cluding cell culture and reagents; screen validation and drug sensitivity assays; A systematic study of kinase-driven EGFR bypass may reveal immunoblotting; gene expression profiling; and LINCS analysis, are described the scope of potential kinases switches, and whether they act in SI Materials and Methods. through common or divergent pathways in sustaining EGFR- independent survival. Our findings suggest that the diverse ACKNOWLEDGMENTS. We thank the Broad Genetic Perturbation Platform kinases capable of replacing EGFR in PC9 cells uniformly for generation of the ORF library; the Broad Connectivity Map team for converge upon downstream pathways. generation of the Library of Integrated Network-Based Cellular Signatures More generally, our observation that a large number of kinase (LINCS) dataset and technical assistance; V. Dancik for LINCS compound identification; and J. Chmielecki, H. Greulich, E. Stover, Z. Tothova, and X. Wu for inputs can redundantly sustain cancer cell survival is consistent helpful discussions. Support for this work was provided by National Cancer In- with recent reports describing broad potential for growth factor- stitute Grants R01CA116020, R01CA109038, and P50CA090578 (to M.M.) and mediated inhibitor resistance in several tumor dependency models National Institutes of Health Common Fund Grant 5U54HG006093 (to T.R.G.).

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