Functional epigenetics approach identifiesBRM/SMARCA2 as a critical synthetic lethaltarget in BRG1-deficient cancersGregory R. Hoffmana, Rami Rahalb,1, Frank Buxtona, Kay Xiangb, Gregory McAllistera, Elizabeth Friasa,Linda Bagdasarianc, Janina Huberb, Alicia Lindemana, Dongshu Chenb, Rodrigo Romerob, Nadire Ramadana,Tanushree Phadkea, Kristy Haasb, Mariela Jaskelioffb, Boris G. Wilsond, Matthew J. Meyerb, Veronica Saenz-Vashb,Huili Zhaib, Vic E. Myera, Jeffery A. Portera, Nicholas Keenb, Margaret E. McLaughlinc, Craig Mickanina,Charles W. M. Robertsd, Frank Stegmeierb,2, and Zainab Jaganib,2

Departments of aDevelopmental and Molecular Pathways and bOncology, Novartis Institutes for BioMedical Research, Cambridge, MA 02139; cOncologyTranslational Medicine, Novartis Pharma, Cambridge, MA 02139; and dDepartment of Pediatric Oncology, Dana–Farber Cancer Institute, Boston, MA 02215

Edited* by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved January 22, 2014 (received for review September 6, 2013)

Defects in epigenetic regulation play a fundamental role in thedevelopment of cancer, and epigenetic regulators have recentlyemerged as promising therapeutic candidates. We therefore setout to systematically interrogate epigenetic cancer dependenciesby screening an epigenome-focused deep-coverage design shRNA(DECODER) library across 58 cancer cell lines. This screen identifiedBRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essentialfor the growth of tumor cells that harbor loss of function mu-tations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficientcancer cells leads to a cell cycle arrest, induction of senescence,and increased levels of global H3K9me3. We further demonstratethe selective dependency of BRG1-mutant tumors on BRM in vivo.Genetic alterations of the mSWI/SNF chromatin remodeling com-plexes are the most frequent among chromatin regulators in can-cers, with BRG1/SMARCA4 mutations occurring in ∼10–15% of lungadenocarcinomas. Our findings position BRM as an attractive ther-apeutic target for BRG1 mutated cancers. Because BRG1 and BRMfunction as mutually exclusive catalytic subunits of the mSWI/SNFcomplex, we propose that such synthetic lethality may be explainedby paralog insufficiency, in which loss of one family member unveilscritical dependence on paralogous subunits. This concept of “cancer-selective paralog dependency” may provide a more general strat-egy for targeting other tumor suppressor lesions/complexes withparalogous subunits.

Epigenetic dysregulation is a well-documented feature of humancancer. Cancer genome sequencing efforts have revealed re-

current somatic mutations in several chromatin regulators, furtherimplying a causal role for altered chromatin states in tumorigen-esis (1). Indeed, one of the most significant findings from cancergenome profiling is the discovery of frequent mutations in varioussubunits of the mammalian SWI/SNF (mSWI/SNF) chromatinremodeling complex (2, 3). The mSWI/SNF complexes consist ofone of two mutually exclusive DNA-dependent ATPases, BRG1/SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependentregulator of chromatin, subfamily a, member 4) or BRM/SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependentregulator of chromatin, subfamily a, member 2), together withcore and accessory subunits that function in mobilizing nucleo-somes to regulate transcription, DNA replication and repair,and higher-order chromosome dynamics (4, 5). Initial insightsinto the role of mSWI/SNF complexes in tumorigenesis arosefrom identification of biallelic inactivation of the core subunitSNF5/SMARCB1/BAF47 in malignant rhabdoid tumors (6) withsubsequent demonstration of its potent tumor suppressor functionin genetically engineered mouse models of Snf5 inactivation (7, 8).Pointing to the broader relevance of mSWI/SNF complexes in can-cers, mutations in the accessory subunits such as ARID1A/BAF250A

have been reported in ovarian clear cell and endometrial carcinomasamong others (9, 10), and PBRM1/BAF180 in clear cell renal cellcarcinomas (11). Mutations and/or loss of expression of the catalyticsubunit BRG1 have been reported predominantly in nonsmall celllung cancers (12–16), but also in others (2, 17, 18). In support of itstumor suppressor function, BRG1 reexpression inhibits the growthof BRG1-mutant/deficient cancer cell lines (19), and Brg1 het-erozygous mice develop mammary carcinomas (20). Notably,BRG1-mutant cancers can have co-occurring mutations in otherkey oncogenic and tumor suppressor lesions, such as KRAS andLKB1, yet tend to lack the targetable EGFR mutations or ALKtranslocations (12), thus pointing toward a critical need for targetedtherapies for these patients.A significant proportion of epigenetic mutations are inacti-

vating and, thus, cannot be directly targeted. We reasoned, how-ever, that these mutations may alter the epigenetic state of cancercells, thereby exposing unique epigenetic vulnerabilities. To testthis idea, we pursued an unbiased approach to screen for epige-netic dependencies by using a deep coverage shRNA pool acrossa panel of human cancer cell lines from the Cancer Cell LineEncyclopedia (CCLE) (21). This screen strikingly revealed BRM

Significance

Mammalian SWI/SNF (mSWI/SNF) alterations are highly preva-lent, now estimated to occur in 20% of cancers. The inactivatingnature of mSWI/SNF mutations presents a challenge for devisingstrategies to target these epigenetic lesions. By performing acomprehensive pooled shRNA screen of the epigenome usinga unique deep coverage design shRNA (DECODER) library acrossa large cancer cell line panel, we identified that BRG1/SMARCA4mutant cancer cells are highly sensitive to BRM/SMARCA2 de-pletion. Our study provides important mechanistic insight intothe BRM/BRG1 synthetic lethal relationship, shows this findingtranslates in vivo, and highlights BRM as a promising therapeutictarget for the treatment BRG1-mutant cancers.

Author contributions: G.R.H., R. Rahal, F.B., M.J., B.G.W., M.J.M., H.Z., V.E.M., J.A.P., N.K.,M.E.M., C.M., C.W.M.R., F.S., and Z.J. designed research; G.R.H., R. Rahal, F.B., K.X., E.F.,L.B., J.H., A.L., D.C., R. Romero, N.R., T.P., K.H., M.J., V.S.-V., and M.E.M. performed re-search; G.R.H. and F.B. contributed new reagents/analytic tools; G.R.H., R. Rahal, K.X.,G.M., E.F., L.B., D.C., K.H., M.J., V.S.-V., H.Z., M.E.M., and Z.J. analyzed data; and G.R.H.,L.B., V.S.-V., F.S., and Z.J. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1Present address: Blueprint Medicines, Cambridge, MA 02142.2To whom correspondence may be addressed. E-mail: zainab.jagani@novartis.com orfrank.stegmeier@novartis.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1316793111/-/DCSupplemental.

3128–3133 | PNAS | February 25, 2014 | vol. 111 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1316793111

as selectively required for the growth of BRG1-mutant cancer cells.We further provide an in-depth mechanistic understanding of thissynthetic lethal relationship including the biochemical charac-terization of the mSWI/SNF complex following BRM knockdownand comprehensive in vivo characterization of BRM depletion inBRG1-wild type vs. mutant lung cancer models. Collectively,these studies identify BRM as a critical and promising therapeutictarget in BRG1-mutant cancers.

ResultsDeep Coverage Pooled shRNA Screening Reveals BRM/SMARCA2 asa Synthetic Lethal Target in BRG1-Mutant Cancers. Although RNAihas proven to be a powerful forward genetics approach, the ro-bustness and reproducibility of RNAi screens has been chal-lenged by the prevalence of off-target effects and inability topredict high-potency shRNAs with good confidence (22). In aneffort to overcome these limitations, we constructed a deep cov-erage design shRNA (DECODER) library (Fig. 1A) to yield higherconfidence hits through extensive shRNA coverage for eachgene. The DECODER epigenome library contained 17 shRNAsper gene against a diverse collection of epigenetic regulators, witha particular focus on druggable classes, including those involved inthe covalent modification of histones, and proteins with readerdomains recognizing histone marks (SI Appendix, Fig. S1A andDataset S1). This library was screened across a panel of 58 cancercell lines from CCLE representing various primary sites and di-verse genetic backgrounds (ref. 21; Dataset S2). The growth im-pact of shRNAs for each cell line was scored by calculating a zscore based on the fold change in representation of each in-dividual shRNA relative to its representation in the startingplasmid pool as measured by next generation sequencing (see SIAppendix, Fig. S1B and Methods and Dataset S2 for a detailed

description of these calculations). In addition to scoring the in-dividual shRNAs, we derived gene level calls from the 17shRNAs for each gene by applying the Redundant siRNAActivity (RSA) algorithm, which calculates gene-centric P values(23). To identify genes whose product is selectively required forgrowth in a subset of cancer lines, we performed k-means clus-tering (24) of the RSA value for each gene to define groups of“sensitive” and “insensitive” cell lines and subsequently rankedhits based on the difference in cluster centers (SI Appendix,Methods). KRAS, which was included in the library as a posi-tive control, emerged as one of the top differential genes fromthis analysis, having strong growth-inhibitory effects only ina subset of the cancer cell lines profiled. As expected, the dif-ferential activity reflected by the KRAS RSA score stronglycorrelated with KRAS mutation status (P = 9.65 × 10−12; Fig. 1Band SI Appendix, Fig. S1C). In contrast to the genotype selectiveactivity of KRAS, some other genes included in this library such asPSMA3, which encodes a proteasome subunit, appeared to bebroadly cytotoxic to all cell lines (SI Appendix, Fig. S1D).Notably, application of the ATARIS algorithm (25), whichprovides a statistical method for identifying shRNAs that sharea common activity profile, revealed that 10 of 17 independentKRAS shRNAs displayed similar antiproliferative profiles(SI Appendix, Fig. S1E). Collectively, the assessment of thesepositive controls demonstrates the robustness of the DECODERlethality screen approach.Intriguingly, the gene with the strongest robust differential

lethal score from this epigenome library screen was BRM, acatalytic subunit of the mSWI/SNF chromatin remodeling com-plexes, ranking even higher than the KRAS positive control (Fig.1C and SI Appendix, Fig. S2A). To identify whether any specificgenetic or molecular feature correlates with sensitivity to BRMdepletion, we performed a systematic interrogation of all fea-tures in the CCLE, including gene expression, copy number,and mutation status to identify features enriched in the sensi-tive cell lines as defined by the k-means clustering for BRM(21). Strikingly, loss-of-function mutations in the mSWI/SNFcatalytic subunit BRG1 strongly correlated with sensitivity toBRM shRNAs (Fig. 2A, P = 2.03 × 10−7). Notably, the ATARISsolution for BRM identified 12 of the 17 BRM shRNAs in thelibrary as showing a similar antiproliferative profile in BRG1-mutant cancer cells, thereby strongly supporting the notion thatthis differential lethality effect is due to on-target rather than off-target activity (SI Appendix, Fig. S2B).BRM and BRG1 are closely related paralogs that function

as mutually exclusive ATP-dependent catalytic subunits of themSWI/SNF complexes (26). Although BRM and BRG1 are sig-nificantly conserved at the protein level, they display overlappingand distinct functions (27–29). The identification of BRM as asynthetic lethal hit in the context of BRG1 mutations raises thepossibility that BRM is substituting for essential functions of themSWI/SNF complex in BRG1-deficient cancer cells and, thus,creating a cancer-selective vulnerability. A prediction of thismodel would be that complete (i.e., homozygous) loss of BRG1should lead to more pronounced BRM dependency comparedwith heterozygous loss of BRG1. Indeed, cancer cell lines withcomplete loss of BRG1 were highly sensitive to BRM shRNAs,whereas BRM shRNAs had little or no impact in cells that wereeither BRG1 wild type or with heterozygous BRG1 mutations(Fig. 2A and Dataset S3). The status of BRG1 protein expressionin BRG1-mutant and wild-type (WT) lines was further confirmedby immunoblotting (Fig. 2B and SI Appendix, Fig. S3). Our dataalso suggest that sensitivity to BRM shRNAs in the BRG1-mutant setting is not only confined to lung cancer (as noted inthe case of A549, H1299, and H838 lung cancer cell lines),which is the predominant indication in which BRG1 mutationshave been reported, but also include ovarian (TYKNU) andliver (SKHEP1) cancer cell lines with BRG1 loss (Fig. 2A and

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Fig. 1. An epigenome-wide pooled shRNA screen identifies BRM as a syn-thetic lethal target in BRG1-mutant cancer cells. (A) A schematic of thescreening workflow for the shRNA screens. (B) Scatter plot showing the nor-malized counts for each shRNA in the epigenome shRNA library in the originalplasmid pool plotted relative to a sample taken after five-population doublingsfrom a KRAS-mutant pancreatic cancer cell line Mia-Paca-2. The 17 shRNAstargeting KRAS are highlighted in purple, illustrating the loss in repre-sentation for the majority of KRAS shRNAs during the time course of theexperiment. The solid line is drawn to indicate no change in counts, whereasthe dotted lines indicated ±1.5-fold change in counts. (C) Ranking for all ele-ments in the epigenome shRNA library are shown highlighting BRM as the top-ranking hit from the screen. Ranks were calculated for each gene from thelibrary based on the difference in the mean log P value calculated by using theRSA statistic for sensitive cell lines relative to insensitive cell lines. The rank forKRAS is highlighted to illustrate the performance of a positive control thatselectively inhibits growth in KRAS-mutant cell lines.

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Dataset S3). Collectively, these findings demonstrate that cellslacking a functional copy of BRG1 become exquisitely de-pendent on residual BRM containing mSWI/SNF complexesfor their survival.

BRM Depletion Selectively Inhibits the Growth of BRG1-Mutant CancerCells. To further examine the impact of BRM depletion on BRG1-deficient cells, we engineered several BRG1-deficient and WT celllines with doxycycline (dox)-inducible shRNA constructs targetingBRM. In all three BRG1-mutant/deficient lung cancer cell linestested (NCI-H838, NCI-H1299, and A549), induction of BRMshRNAs produced highly efficient depletion of BRM proteinand led to profound growth inhibition in short-term proliferationand colony formation assays (Fig. 3 A, C, and E and SI Appendix,Fig. S4). Consistent with the results from the screening data,BRM knockdown with the same shRNAs that impacted growthin BRG1-mutant cancer cells, did not affect proliferation of cellswith intact BRG1, such as the BRG1-WT lung cancer cell lineNCI-H460 (Fig. 3 B, D, and F) and BEAS2B, a nontumorigenicimmortalized lung epithelial cell line (Fig. 4 A and B and SIAppendix, Fig. S5). When we examined the effects of BRM de-pletion in CORL23 lung cancer cells, which harbor a heterozy-gous BRG1 lesion, we detected a modest impact on cell growth(SI Appendix, Fig. S6). Although this growth inhibitory effect wassignificantly less pronounced than in cells with homozygous loss-of-function BRG1 mutations, these findings raise the interestingpossibility that heterozygous loss of BRG1 may already partially

sensitize cells to BRM inhibition. Alternatively, this heterozy-gous BRG1 mutation, which results in an in-frame deletion, maylead to mild dominant negative effects. To further investigate theobserved synthetic lethality, we tested whether expression of eitherBRM or BRG1 is sufficient to sustain cancer cell proliferation andwhether cells can tolerate combined inactivation of BRM andBRG1. Indeed, although depletion of BRM or BRG1 did notimpact the proliferation of two BRG1-WT cell lines, simultaneousknockdown of BRG1 and BRM led to marked growth inhibitionin both of these BRG1-WT cell lines, strongly supporting thesynthetic lethal relationship between BRM and BRG1 (Fig. 4 A–Dand SI Appendix, Fig. S5).

BRM Knockdown Does Not Disrupt Association of Other Core andAccessory mSWI/SNF Subunits. BRM and BRG1 are catalytic com-ponents and part of the core mSWI/SNF complex. We thereforesought to ascertain the impact of BRG1 mutations and BRMdepletion on mSWI/SNF complex composition and stability. Pu-rification of the mSWI/SNF complex via coimmunoprecipitationof core subunits such as SNF5 or BAF155, and size-exclusionchromatographic separation, showed that a subcomplex contain-ing core and accessory subunits remained intact in BRG1-deficientcells (Fig. 5A and SI Appendix, Figs. S7 and S8). Moreover,knockdown of BRM in BRG1-mutant cancer cells did not appearto destabilize the complex (Fig. 5A and SI Appendix, Figs. S7 andS8). Although the majority of the complex stays intact, we notedthat BAF53A no longer associates with the complex after BRM

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Fig. 2. Complete loss of BRG1 and retention of BRM define the growthinhibitory response of cancer cells to BRM-targeting shRNAs. (A) Waterfallplot showing the log P value calculated with the RSA statistic for BRMshRNAs as in Fig. 1C and colored by BRG1 mutation status (i.e., homozygous,heterozygous, dual loss of BRG1/BRM). (B) Western blot of representativeBRG1-WT and mutant cell lines from the screen showing BRG1 and BRMexpression. VINCULIN is included as a loading control. BRG1-WT cell linesretain BRG1 expression, whereas BRG1 homozygous mutant cell lines sen-sitive to BRM shRNAs (denoted as +) lack BRG1 expression but retain BRMexpression.

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Fig. 3. BRM depletion significantly and selectively inhibits the growth ofBRG1-mutant cancer cells. (A) Western blot showing reduction of BRMprotein upon dox treatment (120 h, 100 ng/mL) of BRG1-mutant/deficientNCI-H838 cells stably transduced with inducible BRM shRNA-2025 or 5537. Anontargeting CTL shRNA was included. (B) Western blot as in A but in BRG1-WTNCI-H460 cells. (C) CTL or BRM shRNA NCI-H838 cells were seeded at 500 cellsper well in a 96-well plate in triplicate. Cells were treated with dox, andcell growth was measured by using the cell titer glo assay at the indicatedtimes. All assays were performed in triplicate, and values are shown asmean ± SD. (D) Cell growth assay as in D but with CTL or BRM shRNA NCI-H460 cells. (E ) CTL or BRM shRNA NCI-H838 cells were seeded at 2,000 cellsper well. Cells were treated with dox (100 ng/mL), and colony formationwas monitored after 11 d with crystal violet staining. (F) CTL or BRM shRNANCI-H460 cells were seeded at 1,000 per well, treated with dox, and monitoredfor colony formation as in E.

3130 | www.pnas.org/cgi/doi/10.1073/pnas.1316793111 Hoffman et al.

depletion (Fig. 5A). Because previous studies have shown thatBAF53A directly interacts with the ATPase subunit of the com-plex (i.e., BRG1) (30), we speculate that BAF53A may interactwith the “residual” BRM ATPase in BRG1-deficient cells, butdissociate from the complex once both ATPase subunits areabsent. Overall, these findings indicate that the observed syntheticlethality cannot simply be explained by destabilization of the entiremSWI/SNF complex, but rather suggests that the specific in-hibition of the redundant activity of BRM and BRG1 suffices toproduce a marked growth defect. To further investigate complexcomposition in the absence of both ATPases, we examined SW13cells, which lack BRG1 and BRM expression. Consistent withprior results, we detected robust association of the core and ac-cessory SWI/SNF subunits, with the exception of BAF53A (SIAppendix, Fig. S9).

BRM Depletion Results in a Growth Arrest and Induction of H3K9me3in BRG1-Mutant Cancer Cells. We next sought to investigate themechanism for growth inhibition in response to BRM depletion.Examination of cell cycle profiles in the BRG1-mutant cell linesNCI-H838 and NCI-H1299 indicated that BRM knockdown ledto a prominent G1 arrest (Fig. 5B and SI Appendix, Fig. S4 C andI) without appearance of a sub-G1 population that would beindicative of cell death. Consistent with these results, we did notobserve any signs of apoptosis upon BRM knockdown, as judgedby Caspase 3 cleavage (SI Appendix, Fig. S10). The G1 arrest wasaccompanied by the appearance of senescent cells as evidencedby flattened cell morphology and positive staining for acidicβ-galactosidase (Fig. 5C and SI Appendix, Fig. S4J), suggestingthat the growth inhibitory effect of BRM is mediated, at least in

this subset of BRM-dependent lines, through induction of G1arrest and senescence. The growth inhibitory effect upon BRMknockdown appears to be irreversible as cells continued to re-main growth arrested even upon withdrawal of dox (SI Appendix,Fig. S11).Given the critical role of the mSWI/SNF complex in chromatin

structure and function, we reasoned that global chromatin pro-filing may provide potential insights toward the molecular mech-anisms associated with the synthetic lethal relationship betweenBRM and BRG1. Using quantitative mass spectrometry-basedmethods, we surveyed a variety of histone modifications in re-sponse to BRM depletion. Although most global histone marksremained unaffected, BRM knockdown induced a significantincrease in H3K9me3 levels in NCI-H1299 cells (SI Appendix,Fig. S12). Immunofluorescence-based detection further confirmedthe substantial increase in H3K9me3 staining upon BRM knock-down (Fig. 5D). Of note, H3K9me3 is a repressive histone markthat is characteristic of heterochromatic gene regions and can beassociated with cells undergoing senescence (31). Thus, themarked increase in repressive H3K9me3 in response to BRMdepletion in BRG1-mutant cells may be reflective of cells enteringa growth arrest/senescent state.

BRM Knockdown Leads to Selective Growth Inhibition of BRG1-MutantTumors in Vivo.The tumor microenvironment can, in some settings,profoundly impact the therapeutic response to chemotherapy andtargeted agents. Hence, we wanted to investigate whether theselective BRM dependency of BRG1-mutant cancers translates invivo. We compared the effects of BRM knockdown in BRG1-mutant NCI-H1299 and BRG1-WT NCI-H460 xenograft models(SI Appendix, Fig. S13A), containing either dox-inducible control(CTL) nontargeting shRNA or two distinct BRM-targeting shRNAs(sh2025 or sh5537). Upon dox treatment, BRM expression was

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Fig. 5. BRM knockdown does not perturb the interaction of core mSWI/SNFsubunits, and leads to a cell cycle arrest and senescence, accompanied byinduction of H3K9me3. (A) Western blot showing detection of mSWI/SNFsubunits upon immunoprecipitation of the core subunit BAF155 or SNF5, inthe absence and presence of dox-induced BRM knockdown in a BRG1-mutant cell line, NCI-H838. (B) BRM shRNA-2025 containing NCI-H838 cellswere treated with or without dox for 7 d and assessed for changes in cell cycleby analysis of DNA content via Propidium Iodide staining. Percentage of cellsdisplaying G1, S, and G2 phase content are shown on each histogram. (C) CTLshRNA or BRM shRNA containing NCI-H838 cells were induced with dox for 7 dand monitored for senescence-associated β-galactosidase staining (blue pre-cipitate). (D) CTL shRNA or BRM shRNA containing NCI-H838 cells were inducedwith dox for 7 d and stained for H3K9me3 and DAPI.

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markedly decreased in the BRM shRNA tumors but not in theCTL shRNA tumors (Fig. 6 A–C and SI Appendix, Fig. S13 B–F). The variability in BRM levels in the CTL shRNA tumorsand dox-treated BRM sh2025 tumors is attributed to intra-tumoral and intertumoral variability in the extent of necrosis(SI Appendix, Fig. S14). Efficient BRM knockdown was main-tained through the end point of the studies (SI Appendix, Fig.S13 B–F). Dox treatment of mice bearing BRG1-mutant NCI-H1299 xenografts with either BRM sh2025 or BRM sh5537 ledto significant inhibition of tumor growth (T/C = 29% and T/C =5%, respectively) (Fig. 6D). This effect was due to depletion ofBRM rather than dox treatment alone, as NCI-H1299 CTLshRNA tumors progressed rapidly despite dox treatment (Fig.6D). BRM depletion led to a marked decrease in the pro-liferation marker Ki67 in dox-treated NCI-H1299 BRM shRNAtumors but not in NCI-H1299 CTL shRNA tumors (Fig. 6 F and

G and SI Appendix, Fig. S15 A and B). Moreover, BRM inhibitionin NCI-H1299 tumors increased expression of the senescencemarker acidic β-gal (SI Appendix, Fig. S16 A and B), but we did notobserve increased apoptosis or an inflammatory response (SIAppendix, Figs. S16 C and D and S17 A–D). Together, thesefindings suggest that similar to the in vitro findings, the in vivogrowth inhibition is mediated by G1 arrest and induction of se-nescence. Importantly, knockdown of BRM did not impact thegrowth of BRG1-WT NCI-H460 tumors (Fig. 6E and SI Appendix,Fig. S15 C and D), demonstrating the selective effects of BRMdepletion in vivo.

DiscussionFunctional genomic approaches, such as pooled shRNA screens,hold great promise for the identification of selective cancer de-pendencies (32, 33). In this study, we used a fundamentally distinctapproach to pooled shRNA screening, relying on DECODER li-braries to increase confidence in hits based on the redundancy ofshRNAs scoring against a target. During the course of our study, asimilar deep-coverage shRNA approach was reported in a screenfor Ricin sensitivity (34). The robustness of hits identified fromthese screens illustrates the power of the DECODER screeningapproach, with the potential to overcome the inherent “noise” inRNAi screening datasets.Our systematic screen for epigenetic dependencies identified

a robust synthetic lethal interaction between BRG1 and BRM.Cancer cells harboring BRG1 mutations are highly sensitive toBRM depletion, demonstrating a unique role for BRM containingcomplexes in promoting tumor cell growth. It is interesting tonote, however, that a subpopulation of lung cancers with BRG1mutations or BRG1 loss are reported to have low/no expression ofBRM (13, 14), suggesting that such cancers have alternatemechanisms that allow survival in the absence of both ATPases.Although it is not known how cancer cells that lose both ATPasessurvive, our data indicates that BRG1-deficient cancer cells ex-pressing BRM remain highly sensitive to BRM inhibition. In fact,we confirmed that BRG1-deficient lines that respond to BRMshRNAs express BRM (Fig. 2B), whereas BRG1-mutant/deficientlung cancer cell lines (SBC-5 and KP4) that have no or barelydetectable expression of BRM (Fig. 2B and SI Appendix, Fig. S3)did not respond to BRM shRNAs (Fig. 2A and Dataset S3). Moredetailed studies of such cancers and preclinical models that sustainproliferation in the absence of BRM and BRG1 will likely provideinsights into potential mechanisms of resistance and informstrategies to prevent and/or combat the emergence of resistance.Our study positions BRM as an attractive therapeutic target in

BRG1-deficient cancers. Although BRM and BRG1 are highlyrelated, they display redundant and distinct roles. Whereas in-activation of BRG1 is embryonic lethal (27), that of BRM resultsin viable animals without any overt deficiencies (28), pointingtoward the potential for a good therapeutic window with BRMselective inhibitors. BRM contains a bromodomain and anATPase domain, thus presenting multiple attractive avenuesfor the development of targeted small molecule inhibitors. Theclinical importance of these findings is highlighted by the prev-alence of BRG1 mutations in several cancers, including lungadenocarcinomas. Of note, previous studies have demonstratedthat SNF5-deficient malignant rhabdoid tumors are selectivelysensitive to BRG1 inhibition (35). Intriguingly, SNF5-deficientmalignant rhabdoid tumors lack expression of BRM (35), there-fore raising the possibility that this synthetic lethality may, in fact,be explained by the codependency of BRG1 on BRM. Further-more, during preparation of this manuscript, another group in-dependently reported similar findings in their study of BRG1/BRM synthetic lethality in nonsmall cell lung cancers (36). Ourdiscovery of BRM as the top hit from a systematic unbiasedscreening approach reinforces the robustness of the BRM/BRG1synthetic lethal relationship. Based on the results presented in this

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Fig. 6. BRM knockdown inhibits the growth of BRG1-mutant tumors invivo. NCI-H1299 cancer cells stably expressing dox-inducible CTL shRNA ortwo distinct BRM-targeting shRNAs (sh2025 or sh5537) were inoculated intomice. Tumor-bearing mice were treated for with either vehicle or dox. (A)Western blot of tumor BRM and VINCULIN (loading control) after 7 d oftreatment. (B) Representative images of BRM IHC staining after 7 d oftreatment. (C) Percentage of nuclei positive for BRM after 7 d of treatment.Graphs represent mean ± SEM (n = 3 per treatment group). (D and E) NCI-H1299 (D) or NCI-H460 (E) cancer cells stably expressing dox-inducible CTL,sh2025, or sh5537 BRM shRNA were inoculated into mice. When tumorvolume reached 100–300 mm3, mice were treated continuously with eithervehicle diet (black circles) or dox-supplemented diet (white circles). The tu-mor volume of vehicle and dox-treated mice is plotted as the mean ± SEM(n = 8 per treatment group). *P < 0.05 of Δ tumor volume for the dox relativeto vehicle-treated group. (F) Representative images of Ki67 IHC staining ofNCI-H1299 tumors after 7-d treatment. (G) Percentage of nuclei positive forKi67 in NCI-H1299 tumors after 7 d of treatment. Graphs represent mean ±SEM (n = 3 per treatment group).

3132 | www.pnas.org/cgi/doi/10.1073/pnas.1316793111 Hoffman et al.

study, we propose a model in which mSWI/SNF mutations lead toa hypomorphic complex that promotes tumorigenesis but cannottolerate complete inactivation. In this setting, BRG1 mutationscreate a cancer-specific vulnerability that can be therapeuticallyexploited by selectively targeting the residual BRM containingcomplex (SI Appendix, Fig. S18). More generally, this modelpredicts that targeting of redundant activities (paralogs) of mu-tated mSWI/SNF subunits may present a broader strategy forblocking the growth of mSWI/SNF-mutated cancers. The find-ings in this study therefore support a general approach fortherapeutic intervention for the large collections of mSWI/SNF-mutated cancers through targeting of the residual mSWI/SNF complex.

Materials and MethodsLibrary Design, Construction, and Screening. A custom 6,500 element shRNAlibrary focused on enzymes involved in epigenetic regulationwas constructedby using chip-based oligonucleotide synthesis and cloned as a pool into thethe pRSI9 lentiviral plasmid (Cellecta). Viral packaging was carried outaccording to the manufacturers recommended protocol. Each cell line wasscreened in duplicate, maintained an average minimal representation of1,000 cells per sRNA, and harvested after five-population doublings. Therepresentation of each barcode in the library was measured by next gen-eration sequencing on an Illumina GA2X. Detailed protocols for the viralpackaging, transduction, screening, and data analysis are provided in SIAppendix, Methods.

Cell Culture, Immunoprecipitation, and Western blotting. NCI-H1299, NCI-H460NCI-H838, and A549 cells were cultured in recommended media. Nuclearlysates were prepared by using the NE-PER Nuclear and Cytoplasmic

Extraction kit (Thermo) by following manufacturer’s recommendations.Immunoprecipitaiton was performed with an anti-BRM antibody (Abcam)and associated SWI/SNF subunits detected by Western blot using standardprotocols. Chemiluminescent signal was detected by using SuperSignal WestFemto Maximum Sensitivity Substrateor Li-Cor Odyssey. Additional detailson methods and antibodies used for immunprecipitation and Westernblotting are provided in SI Appendix, Methods.

Functional Characterization of BRM Knockdown Using Inducible shRNA Constructs.shRNA sequences targeting BRM cloned into the pLKO-Tet-On induciblevector system. The sequences of the oligonucleotides used and details on thecloning and lentiviral production are provided in SI Appendix, Methods. Lungcancer cell lines were infected with lentiviruses carrying BRM shRNAs, andthe effect of BRM knockdown in growth and focus formation assays wasdetermined in the presence or absence of dox-induced shRNA expression.Details for the cell cycle, senescence, and immunofluorescence assays areprovided in SI Appendix, Methods.

In Vivo Efficacy Studies. All animal studies were carried out according to theNovartis Guide for the Care and Use of Laboratory Animals. Mice were in-oculated s.c. with NCI-H1299 or NCI-H460 cancer cells stably expressing dox-inducible CTL nontargeting shRNA or two distinct BRM-targeting shRNAs,and tumor volume was measured twice weekly. At termination of eachstudy, tumor tissue was collected from each group and processed for im-munohistochemistry by using standard methods. A detailed description ofthese xenograft and the immunohistochemistry (IHC) studies is provided inSI Appendix, Methods.

ACKNOWLEDGMENTS.We thank Bill Forrester and Nathan Ross for discussionsand a critical reading of the manuscript.

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