Functional Epigenetics Approach Identifies BRM/SMARCA2 As a Critical Synthetic Lethal Target in BRG1-Deficient Cancers

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers

Gregory 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; cOncology Translational 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 the have been reported in ovarian clear cell and endometrial carcinomas development of cancer, and epigenetic regulators have recently among others (9, 10), and PBRM1/BAF180 in clear cell renal cell emerged as promising therapeutic candidates. We therefore set carcinomas (11). Mutations and/or loss of expression of the catalytic out to systematically interrogate epigenetic cancer dependencies subunit BRG1 have been reported predominantly in nonsmall cell by screening an epigenome-focused deep-coverage design shRNA lung cancers (12–16), but also in others (2, 17, 18). In support of its (DECODER) library across 58 cancer cell lines. This screen identified tumor suppressor function, BRG1 reexpression inhibits the growth BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/ of BRG1-mutant/deficient cancer cell lines (19), and Brg1 het- SNF (mSWI/SNF) chromatin remodeling complex, as being essential erozygous mice develop mammary carcinomas (20). Notably, for the growth of tumor cells that harbor loss of function mu- BRG1-mutant cancers can have co-occurring mutations in other BRG1/SMARCA4. tations in Depletion of BRM in BRG1-deficient key oncogenic and tumor suppressor lesions, such as KRAS and cancer cells leads to a cell cycle arrest, induction of senescence, LKB1, yet tend to lack the targetable EGFR mutations or ALK and increased levels of global H3K9me3. We further demonstrate translocations (12), thus pointing toward a critical need for targeted the selective dependency of BRG1-mutant tumors on BRM in vivo. therapies for these patients. Genetic alterations of the mSWI/SNF chromatin remodeling com- A significant proportion of epigenetic mutations are inacti- plexes are the most frequent among chromatin regulators in can- vating and, thus, cannot be directly targeted. We reasoned, how- cers, with BRG1/SMARCA4 mutations occurring in ∼10–15% of lung ever, that these mutations may alter the epigenetic state of cancer adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM cells, thereby exposing unique epigenetic vulnerabilities. To test function as mutually exclusive catalytic subunits of the mSWI/SNF this idea, we pursued an unbiased approach to screen for epige- complex, we propose that such synthetic lethality may be explained netic dependencies by using a deep coverage shRNA pool across by paralog insufficiency, in which loss of one family member unveils a panel of human cancer cell lines from the Cancer Cell Line critical dependence on paralogous subunits. This concept of “cancer- Encyclopedia (CCLE) (21). This screen strikingly revealed BRM selective paralog dependency” may provide a more general strategy for targeting other tumor suppressor lesions/complexes with Significance paralogous subunits. Mammalian SWI/SNF (mSWI/SNF) alterations are highly preva- pigenetic dysregulation is a well-documented feature of human lent, now estimated to occur in 20% of cancers. The inactivating Ecancer. Cancer genome sequencing efforts have revealed re- nature of mSWI/SNF mutations presents a challenge for devising current somatic mutations in several chromatin regulators, further strategies to target these epigenetic lesions. By performing a implying a causal role for altered chromatin states in tumorigen- comprehensive pooled shRNA screen of the epigenome using esis (1). Indeed, one of the most significant findings from cancer a unique deep coverage design shRNA (DECODER) library across BRG1/SMARCA4 genome profiling is the discovery of frequent mutations in various a large cancer cell line panel, we identified that BRM/SMARCA2 subunits of the mammalian SWI/SNF (mSWI/SNF) chromatin mutant cancer cells are highly sensitive to de- remodeling complex (2, 3). The mSWI/SNF complexes consist of pletion. Our study provides important mechanistic insight into one of two mutually exclusive DNA-dependent ATPases, BRG1/ the BRM/BRG1 synthetic lethal relationship, shows this finding translates in vivo, and highlights BRM as a promising therapeutic SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent target for the treatment BRG1-mutant cancers. regulator of chromatin, subfamily a, member 4) or BRM/

SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent 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., regulator of chromatin, subfamily a, member 2), together with 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., core and accessory subunits that function in mobilizing nucleo- 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- somes to regulate transcription, DNA replication and repair, 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., and higher-order chromosome dynamics (4, 5). Initial insights L.B., V.S.-V., F.S., and Z.J. wrote the paper. into the role of mSWI/SNF complexes in tumorigenesis arose The authors declare no conflict of interest. from identification of biallelic inactivation of the core subunit *This Direct Submission article had a prearranged editor. SNF5/SMARCB1/BAF47 in malignant rhabdoid tumors (6) with 1Present address: Blueprint Medicines, Cambridge, MA 02142. subsequent demonstration of its potent tumor suppressor function 2To whom correspondence may be addressed. E-mail: [email protected] or in genetically engineered mouse models of Snf5 inactivation (7, 8). [email protected]. Pointing to the broader relevance of mSWI/SNF complexes in can- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cers, mutations in the accessory subunits such as ARID1A/BAF250A 1073/pnas.1316793111/-/DCSupplemental.

3128–3133 | PNAS | February 25, 2014 | vol. 111 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1316793111 Downloaded by guest on September 27, 2021 as selectively required for the growth of BRG1-mutant cancer cells. description of these calculations). In addition to scoring the in- We further provide an in-depth mechanistic understanding of this dividual shRNAs, we derived gene level calls from the 17 synthetic lethal relationship including the biochemical charac- shRNAs for each gene by applying the Redundant siRNA terization of the mSWI/SNF complex following BRM knockdown Activity (RSA) algorithm, which calculates gene-centric P values and comprehensive in vivo characterization of BRM depletion in (23). To identify genes whose product is selectively required for BRG1-wild type vs. mutant lung cancer models. Collectively, growth in a subset of cancer lines, we performed k-means clus- these studies identify BRM as a critical and promising therapeutic tering (24) of the RSA value for each gene to define groups of “ ” “ ” target in BRG1-mutant cancers. sensitive and insensitive cell lines and subsequently ranked hits based on the difference in cluster centers (SI Appendix, Results Methods). KRAS, which was included in the library as a posi- Deep Coverage Pooled shRNA Screening Reveals BRM/SMARCA2 as tive control, emerged as one of the top differential genes from a Synthetic Lethal Target in BRG1-Mutant Cancers. Although RNAi this analysis, having strong growth-inhibitory effects only in has proven to be a powerful forward genetics approach, the ro- a subset of the cancer cell lines profiled. As expected, the differential activity reflected by the KRAS RSA score strongly bustness and reproducibility of RNAi screens has been chal- − correlated with KRAS mutation status (P = 9.65 × 10 12;Fig.1B lenged by the prevalence of off-target effects and inability to SI Appendix C predict high-potency shRNAs with good confidence (22). In an and , Fig. S1 ). In contrast to the genotype selective effort to overcome these limitations, we constructed a deep cov- activity of KRAS, some other genes included in this library such as PSMA3, which encodes a proteasome subunit, appeared to be erage design shRNA (DECODER) library (Fig. 1A)toyieldhigher broadly cytotoxic to all cell lines (SI Appendix,Fig.S1D). confidence hits through extensive shRNA coverage for each Notably, application of the ATARIS algorithm (25), which gene. The DECODER epigenome library contained 17 shRNAs provides a statistical method for identifying shRNAs that share per gene against a diverse collection of epigenetic regulators, with a common activity profile, revealed that 10 of 17 independent a particular focus on druggable classes, including those involved in KRAS shRNAs displayed similar antiproliferative profiles the covalent modification of histones, and proteins with reader (SI Appendix,Fig.S1E). Collectively, the assessment of these SI Appendix A domains recognizing histone marks ( ,Fig.S1 and positive controls demonstrates the robustness of the DECODER Dataset S1). This library was screened across a panel of 58 cancer lethality screen approach. cell lines from CCLE representing various primary sites and di- Intriguingly, the gene with the strongest robust differential verse genetic backgrounds (ref. 21; Dataset S2). The growth im- lethal score from this epigenome library screen was BRM, a pact of shRNAs for each cell line was scored by calculating a z catalytic subunit of the mSWI/SNF chromatin remodeling com- score based on the fold change in representation of each in- plexes, ranking even higher than the KRAS positive control (Fig. dividual shRNA relative to its representation in the starting 1C and SI Appendix, Fig. S2A). To identify whether any specific plasmid pool as measured by next generation sequencing (see SI genetic or molecular feature correlates with sensitivity to BRM Appendix, Fig. S1B and Methods and Dataset S2 for a detailed depletion, we performed a systematic interrogation of all features in the CCLE, including gene expression, copy number, andmutationstatustoidentifyfeaturesenrichedinthesensi- tive cell lines as defined by the k-means clustering for BRM A infect harvest NGS chip based plasmid lentivirus cells cells (21). Strikingly, loss-of-function mutations in the mSWI/SNF oligo synthesis pool pool catalytic subunit BRG1 strongly correlated with sensitivity to − BRM shRNAs (Fig. 2A, P = 2.03 × 10 7). Notably, the ATARIS solution for BRM identified 12 of the 17 BRM shRNAs in the 5-doublings library as showing a similar antiproliferative profile in BRG1- 17 shRNAs mutant cancer cells, thereby strongly supporting the notion that per gene B C this differential lethality effect is due to on-target rather than off- BRM target activity (SI Appendix, Fig. S2B). 10000 10 KRAS BRM and BRG1 are closely related paralogs that function KRAS 1000 8 as mutually exclusive ATP-dependent catalytic subunits of the mSWI/SNF complexes (26). Although BRM and BRG1 are sig- 6 100

Rank nificantly conserved at the protein level, they display overlapping 4 and distinct functions (27–29). The identification of BRM as a 10 BRG1 2 synthetic lethal hit in the context of mutations raises the

5-doublings Counts 1 possibility that BRM is substituting for essential functions of the 0 1 10 100 1000 10000 Gene mSWI/SNF complex in BRG1-deficient cancer cells and, thus, Plasmid Counts creating a cancer-selective vulnerability. A prediction of this model would be that complete (i.e., homozygous) loss of BRG1 Fig. 1. An epigenome-wide pooled shRNA screen identifies BRM as a synthetic lethal target in BRG1-mutant cancer cells. (A) A schematic of the should lead to more pronounced BRM dependency compared screening workflow for the shRNA screens. (B) Scatter plot showing the nor- with heterozygous loss of BRG1. Indeed, cancer cell lines with malized counts for each shRNA in the epigenome shRNA library in the original complete loss of BRG1 were highly sensitive to BRM shRNAs, plasmid pool plotted relative to a sample taken after five-population doublings whereas BRM shRNAs had little or no impact in cells that were from a KRAS-mutant pancreatic cancer cell line Mia-Paca-2. The 17 shRNAs either BRG1 wild type or with heterozygous BRG1 mutations targeting KRAS are highlighted in purple, illustrating the loss in repre- (Fig. 2A and Dataset S3). The status of BRG1 protein expression MEDICAL SCIENCES sentation for the majority of KRAS shRNAs during the time course of the in BRG1-mutant and wild-type (WT) lines was further confirmed experiment. The solid line is drawn to indicate no change in counts, whereas by immunoblotting (Fig. 2B and SI Appendix,Fig.S3). Our data ± the dotted lines indicated 1.5-fold change in counts. (C) Ranking for all ele- also suggest that sensitivity to BRM shRNAs in the BRG1- 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 the mutant setting is not only confined to lung cancer (as noted in the case of A549, H1299, and H838 lung cancer cell lines), library based on the difference in the mean log P value calculated by using the BRG1 RSA statistic for sensitive cell lines relative to insensitive cell lines. The rank for which is the predominant indication in which mutations KRAS is highlighted to illustrate the performance of a positive control that have been reported, but also include ovarian (TYKNU) and selectively inhibits growth in KRAS-mutant cell lines. liver (SKHEP1) cancer cell lines with BRG1 loss (Fig. 2A and

Hoffman et al. PNAS | February 25, 2014 | vol. 111 | no. 8 | 3129 Downloaded by guest on September 27, 2021 A sensitize cells to BRM inhibition. Alternatively, this heterozy- 0 gous BRG1 mutation, which results in an in-frame deletion, may -2 lead to mild dominant negative effects. To further investigate the observed synthetic lethality, we tested whether expression of either -4 BRG1 homozygous loss BRM or BRG1 is sufficient to sustain cancer cell proliferation and -6 Lacks BRG1 & BRM Expression whether cells can tolerate combined inactivation of BRM and BRG1 Heterozygous -8 BRG1. Indeed, although depletion of BRM or BRG1 did not No genetic data impact the proliferation of two BRG1-WT cell lines, simultaneous WT -10 knockdown of BRG1 and BRM led to marked growth inhibition

Log P-Value (RSA) Log P-Value -12 in both of these BRG1-WT cell lines, strongly supporting the A–D -14 synthetic lethal relationship between BRM and BRG1 (Fig. 4 and SI Appendix,Fig.S5). H4 RD KP4 RKO LK-2 ES-2 10 A549 GP2d A-498 HT-29 786-O G-401 HPAC PC-14 JHH-7 HuH-7 A2780 A2058 HMCB SBC-5 OVISE Hep3B RMG-1 BT-549 SW480 Hey-A8 TYK-nu KNS-62 KP-1NL HCC-44 HCC-15 JHOS-2 HGC-27 JMSU-1 LS411N HCT116 JHOM-1 RMUG-S HMC-1-8 HEC-151 SNU-449 Ishikawa COR-L23 TOV-21G HEC-50B OVTOKO JHUEM-1 HCC1806 KMRC-20 NCI-H838 U-251 MG SK-HEP-1 KYSE-150 BRM Knockdown Does Not Disrupt Association of Other Core and NCI-H1299 LCLC-103H MIA PaCa-2 VMRC-RCW Pa-Tu-8988T Cell Line Accessory mSWI/SNF Subunits. BRM and BRG1 are catalytic com- ponents and part of the core mSWI/SNF complex. We therefore B BRG1/ sought to ascertain the impact of BRG1 mutations and BRM BRG1 Mut. BRG1 Mut. BRM depletion on mSWI/SNF complex composition and stability. Pu- BRG1 WT (Hom) (Het) Loss rification of the mSWI/SNF complex via coimmunoprecipitation of core subunits such as SNF5 or BAF155, and size-exclusion chromatographic separation, showed that a subcomplex containing core and accessory subunits remained intact in BRG1-deficient cells (Fig. 5A and SI Appendix,Figs.S7andS8). Moreover, KP4 A2780 TYKNU A549 HEC151 LCLC103H HCC44 SBC5 SKHEP1 CORL23 H838 NCI -H460 KNS62 HCC15 H1299 BRG1 BRG1 knockdown of BRM in -mutant cancer cells did not appear to destabilize the complex (Fig. 5A and SI Appendix, Figs. S7 and BRM S8). Although the majority of the complex stays intact, we noted Vinculin that BAF53A no longer associates with the complex after BRM --- -++++++-- ---Sensitive

Fig. 2. Complete loss of BRG1 and retention of BRM define the growth inhibitory response of cancer cells to BRM-targeting shRNAs. (A) Waterfall A B plot showing the log P value calculated with the RSA statistic for BRM H838 (BRG1 I873 Splice) H460 (BRG1 Wild Type) shRNAs as in Fig. 1C and colored by BRG1 mutation status (i.e., homozygous, CTL BRM BRM CTL BRM BRM shRNA sh-2025 sh-5537 shRNA sh-2025 sh-5537 heterozygous, dual loss of BRG1/BRM). (B) Western blot of representative -+-+-+Dox -+-+-+Dox BRG1-WT and mutant cell lines from the screen showing BRG1 and BRM BRM BRM expression. VINCULIN is included as a loading control. BRG1-WT cell lines β-TUBULIN β-TUBULIN retain BRG1 expression, whereas BRG1 homozygous mutant cell lines sensitive to BRM shRNAs (denoted as +) lack BRG1 expression but retain BRM C D expression. NCI-H838 NCI-H460 CTL BRM BRM CTL BRM BRM shRNA sh-2025 sh-5537 shRNA sh-2025 sh-5537 3 3 100 100 Dataset S3). Collectively, these findings demonstrate that cells 100 200 200 80 80 80 200 +Dox +Dox 150 lacking a functional copy of BRG1 become exquisitely de- 60 60 60 150 150 40 40 40 100 100 100 pendent on residual BRM containing mSWI/SNF complexes 20 20 50 50 50 LUM (x10 ) LUM (x10 ) 20 -Dox for their survival. 0 0 0 -Dox 0 0 0 0 4 7 0 4 7 0 4 7 0 4 7 0 4 7 0 4 7 BRG1 Day Day Day Day Day Day BRM Depletion Selectively Inhibits the Growth of -Mutant Cancer E F Cells. To further examine the impact of BRM depletion on BRG1- NCI-H838 NCI-H460 deficient cells, we engineered several BRG1-deficient and WT cell CTL BRM BRM CTL BRM BRM shRNA sh-2025 sh-5537 shRNA sh-2025 sh-5537 O +DOX - DOX lines with doxycycline (dox)-inducible shRNA constructs targeting - DOX BRM. In all three BRG1-mutant/deficient lung cancer cell lines tested (NCI-H838, NCI-H1299, and A549), induction of BRM Day 11 Day Day 11 Day shRNAs produced highly efficient depletion of BRM protein +DOX and led to profound growth inhibition in short-term proliferation and colony formation assays (Fig. 3 A, C, and E and SI Appendix, Fig. S4). Consistent with the results from the screening data, Fig. 3. BRM depletion significantly and selectively inhibits the growth of BRM knockdown with the same shRNAs that impacted growth BRG1-mutant cancer cells. (A) Western blot showing reduction of BRM in BRG1-mutant cancer cells, did not affect proliferation of cells protein upon dox treatment (120 h, 100 ng/mL) of BRG1-mutant/deficient NCI-H838 cells stably transduced with inducible BRM shRNA-2025 or 5537. A with intact BRG1, such as the BRG1-WT lung cancer cell line B D F nontargeting CTL shRNA was included. (B) Western blot as in A but in BRG1-WT NCI-H460 (Fig. 3 , ,and ) and BEAS2B, a nontumorigenic NCI-H460 cells. (C) CTL or BRM shRNA NCI-H838 cells were seeded at 500 cells immortalized lung epithelial cell line (Fig. 4 A and B and SI per well in a 96-well plate in triplicate. Cells were treated with dox, and Appendix, Fig. S5). When we examined the effects of BRM de- cell growth was measured by using the cell titer glo assay at the indicated pletion in CORL23 lung cancer cells, which harbor a heterozy- times. All assays were performed in triplicate, and values are shown as gous BRG1 lesion, we detected a modest impact on cell growth mean ± SD. (D) Cell growth assay as in D but with CTL or BRM shRNA NCI- (SI Appendix, Fig. S6). Although this growth inhibitory effect was H460 cells. (E) CTL or BRM shRNA NCI-H838 cells were seeded at 2,000 cells per well. Cells were treated with dox (100 ng/mL), and colony formation significantly less pronounced than in cells with homozygous loss- was monitored after 11 d with crystal violet staining. (F) CTL or BRM shRNA of-function BRG1 mutations, these findings raise the interesting NCI-H460 cells were seeded at 1,000 per well, treated with dox, and monitored possibility that heterozygous loss of BRG1 may already partially for colony formation as in E.

3130 | www.pnas.org/cgi/doi/10.1073/pnas.1316793111 Hoffman et al. Downloaded by guest on September 27, 2021 this subset of BRM-dependent lines, through induction of G A BEAS2B (BRG1 Wild-type) B BEAS2B (BRG1 Wild-type) 1 arrest and senescence. The growth inhibitory effect upon BRM knockdown appears to be irreversible as cells continued to re- CTL BRG1 BRM CTL BRG1 BRM SI Appendix shRNA sh-2202 sh-2025 Dual shRNA sh-2202 sh-2025 Dual main growth arrested even upon withdrawal of dox ( , -+-+ -+-+Dox Fig. S11). BRG1 Given the critical role of the mSWI/SNF complex in chromatin BRM structure and function, we reasoned that global chromatin pro-

β-ACTIN filing may provide potential insights toward the molecular mechanisms associated with the synthetic lethal relationship between C H460 (BRG1 Wild-type) D H460 (BRG1 Wild-type) BRM and BRG1. Using quantitative mass spectrometry-based

1.2 methods, we surveyed a variety of histone modifications in re- 1.0 d11 sponse to BRM depletion. Although most global histone marks CTL BRG1 BRM 0.8 d16 remained unaffected, BRM knockdown induced a significant shRNA sh-2202 sh-2025 Dual 0.6 -+-+-+-+Dox increase in H3K9me3 levels in NCI-H1299 cells (SI Appendix, 0.4 BRG1 0.2 Fig. S12). Immunofluorescence-based detection further confirmed BRM 0.0 the substantial increase in H3K9me3 staining upon BRM knock- Ratio to (relative -dox) -+-+-+-+Dox BRG1 BRM down (Fig. 5D). Of note, H3K9me3 is a repressive histone mark β-TUBULIN NTC Dual sh-2202 sh-2025 that is characteristic of heterochromatic gene regions and can be Fig. 4. Dual but not sole BRG1 and BRM knockdown inhibits the growth of associated with cells undergoing senescence (31). Thus, the BRG1 WT cells. (A) Western blot for BRG1 and BRM levels in lysates from CTL marked increase in repressive H3K9me3 in response to BRM shRNA, BRG1 shRNA-2202, BRM shRNA-2025, or dual (BRG1 shRNA-2202 and depletion in BRG1-mutant cells may be reflective of cells entering BRM shRNA-2025) shRNA containing BEAS2B cells (nontransformed/ im- a growth arrest/senescent state. mortalized) that were treated for 3 d with or without dox. β-Actin was used a loading control. (B) CTL, BRG1 shRNA2202, BRM shRNA-2025, or dual BRM Knockdown Leads to Selective Growth Inhibition of BRG1-Mutant (BRG1 shRNA-2202 and BRM shRNA-2025) shRNA containing BEAS2B cells Tumors in Vivo. The tumor microenvironment can, in some settings, were seeded at 500 cells per well and treated with or without dox for 10 d. Colony formation was monitored with crystal violet staining. (C) Western profoundly impact the therapeutic response to chemotherapy and blot for BRG1 and BRM levels in lysates from CTL shRNA, BRG1 shRNA-2202, targeted agents. Hence, we wanted to investigate whether the BRM shRNA-2025, or dual (BRG1 shRNA-2202 and BRM shRNA-2025) shRNA selective BRM dependency of BRG1-mutant cancers translates in containing BRG1 WT NCI-H460 lung cancer cells that were treated for 3 d vivo. We compared the effects of BRM knockdown in BRG1- with or without dox. β-Tubulin was used a loading control. (D) CTL, BRG1 mutant NCI-H1299 and BRG1-WT NCI-H460 xenograft models shRNA-2202, BRM shRNA-2025, or dual (BRG1 shRNA-2202 and BRM shRNA- (SI Appendix,Fig.S13A), containing either dox-inducible control 2025) shRNA containing BRG1 WT NCI-H460 lung cancer cells were seeded in (CTL) nontargeting shRNA or two distinct BRM-targeting shRNAs six-well plates and treated for 11 or 16 d with or without dox. Cell number was quantified by a Trypan blue exclusion assay and normalized to the –dox (sh2025 or sh5537). Upon dox treatment, BRM expression was sample for each cell line. Experiment shown is representative of three independent experiments.

A IP antibody B 600 shRNA CTL A Input BAF155 SNF5 IgG 400 41% G1 45% G1 depletion (Fig. 5 ). Because previous studies have shown that Dox -+-+-+-+ 38% S 400 35% S 18% G2 BRM 200 Count 17% G2 BAF53A directly interacts with the ATPase subunit of the com- Count 200 plex (i.e., BRG1) (30), we speculate that BAF53A may interact BAF170 “ ” BAF155 400 800 400 800 with the residual BRM ATPase in BRG1-deficient cells, but PI-A PI-A

BAF47 BRM sh-2025 dissociate from the complex once both ATPase subunits are 600 BAF60B 45% G1 75% G1 400 absent. Overall, these findings indicate that the observed synthetic 400 36% S 14% S BAF250 * 16% G2 6% G2 Count lethality cannot simply be explained by destabilization of the entire BAF57 200 Count 200 mSWI/SNF complex, but rather suggests that the specific in- BAF250B hibition of the redundant activity of BRM and BRG1 suffices to 400 800 400 800 BAF53A PI-A PI-A produce a marked growth defect. To further investigate complex D composition in the absence of both ATPases, we examined SW13 C -Dox +Dox cells, which lack BRG1 and BRM expression. Consistent with -Dox +Dox prior results, we detected robust association of the core and ac- H3K9me3 DAPI CTL SI CTL shRNA cessory SWI/SNF subunits, with the exception of BAF53A ( shRNA Appendix,Fig.S9).

BRM Depletion Results in a Growth Arrest and Induction of H3K9me3 BRM BRM sh-2025 in BRG1-Mutant Cancer Cells. We next sought to investigate the sh-2025 mechanism for growth inhibition in response to BRM depletion. BRG1 Fig. 5. BRM knockdown does not perturb the interaction of core mSWI/SNF Examination of cell cycle profiles in the -mutant cell lines subunits, and leads to a cell cycle arrest and senescence, accompanied by NCI-H838 and NCI-H1299 indicated that BRM knockdown led induction of H3K9me3. (A) Western blot showing detection of mSWI/SNF to a prominent G1 arrest (Fig. 5B and SI Appendix, Fig. S4 C and subunits upon immunoprecipitation of the core subunit BAF155 or SNF5, in MEDICAL SCIENCES I) without appearance of a sub-G1 population that would be the absence and presence of dox-induced BRM knockdown in a BRG1- indicative of cell death. Consistent with these results, we did not mutant cell line, NCI-H838. (B) BRM shRNA-2025 containing NCI-H838 cells observe any signs of apoptosis upon BRM knockdown, as judged were treated with or without dox for 7 d and assessed for changes in cell cycle by analysis of DNA content via Propidium Iodide staining. Percentage of cells by Caspase 3 cleavage (SI Appendix, Fig. S10). The G1 arrest was displaying G1,S,andG2 phase content are shown on each histogram. (C)CTL accompanied by the appearance of senescent cells as evidenced shRNA or BRM shRNA containing NCI-H838 cells were induced with dox for 7 d by flattened cell morphology and positive staining for acidic and monitored for senescence-associated β-galactosidase staining (blue pre- β-galactosidase (Fig. 5C and SI Appendix, Fig. S4J), suggesting cipitate). (D) CTL shRNA or BRM shRNA containing NCI-H838 cells were induced that the growth inhibitory effect of BRM is mediated, at least in with dox for 7 d and stained for H3K9me3 and DAPI.

Hoffman et al. PNAS | February 25, 2014 | vol. 111 | no. 8 | 3131 Downloaded by guest on September 27, 2021 markedly decreased in the BRM shRNA tumors but not in the G and SI Appendix,Fig.S15A and B). Moreover, BRM inhibition CTL shRNA tumors (Fig. 6 A–C and SI Appendix,Fig.S13B– in NCI-H1299 tumors increased expression of the senescence F). The variability in BRM levels in the CTL shRNA tumors marker acidic β-gal (SI Appendix,Fig.S16A and B), but we did not and dox-treated BRM sh2025 tumors is attributed to intra- observe increased apoptosis or an inflammatory response (SI tumoral and intertumoral variability in the extent of necrosis Appendix, Figs. S16 C and D and S17 A–D). Together, these (SI Appendix,Fig.S14). Efficient BRM knockdown was main- findings suggest that similar to the in vitro findings, the in vivo tained through the end point of the studies (SI Appendix,Fig. growth inhibition is mediated by G1 arrest and induction of se- B–F BRG1 nescence. Importantly, knockdown of BRM did not impact the S13 ). Dox treatment of mice bearing -mutant NCI- BRG1 E SI Appendix H1299 xenografts with either BRM sh2025 or BRM sh5537 led growth of -WT NCI-H460 tumors (Fig. 6 and , Fig. S15 C and D), demonstrating the selective effects of BRM to significant inhibition of tumor growth (T/C = 29% and T/C = depletion in vivo. 5%, respectively) (Fig. 6D). This effect was due to depletion of BRM rather than dox treatment alone, as NCI-H1299 CTL Discussion shRNA tumors progressed rapidly despite dox treatment (Fig. Functional genomic approaches, such as pooled shRNA screens, D 6 ). BRM depletion led to a marked decrease in the pro- hold great promise for the identification of selective cancer de- BRM liferation marker Ki67 in dox-treated NCI-H1299 shRNA pendencies (32, 33). In this study, we used a fundamentally distinct tumors but not in NCI-H1299 CTL shRNA tumors (Fig. 6 F and approach to pooled shRNA screening, relying on DECODER li- braries to increase confidence in hits based on the redundancy of shRNAs scoring against a target. During the course of our study, a A CTL sh2025 sh5537 similar deep-coverage shRNA approach was reported in a screen Dox: -++ -+- for Ricin sensitivity (34). The robustness of hits identified from BRM these screens illustrates the power of the DECODER screening Vinculin approach, with the potential to overcome the inherent “noise” in RNAi screening datasets. 100 B CTL sh2025 sh5537 C ns *** *** Our systematic screen for epigenetic dependencies identified 80 a robust synthetic lethal interaction between BRG1 and BRM.

60 Cancer cells harboring BRG1 mutations are highly sensitive to -Dox 40 BRM depletion, demonstrating a unique role for BRM containing nuclei BRM % BRM+ 20 complexes in promoting tumor cell growth. It is interesting to NCI-H1299 BRG1 +Dox note, however, that a subpopulation of lung cancers with Dox +- +- +- CTL sh2025 sh5537 mutations or BRG1 loss are reported to have low/no expression of D BRM (13, 14), suggesting that such cancers have alternate 1750 CTL 1750 sh2025 1750 sh5537 1250 1250 1250 mechanisms that allow survival in the absence of both ATPases. ns -Dox 750 750 750 Although it is not known how cancer cells that lose both ATPases *** +Dox (mm³) *** Tumor Tumor

Volume Volume 250 250 250 survive, our data indicates that BRG1-deficient cancer cells ex- NCI-H1299 14 20 26 32 14 20 26 32 14 20 26 32 pressing BRM remain highly sensitive to BRM inhibition. In fact, Days Days Days we confirmed that BRG1-deficient lines that respond to BRM B BRG1 E 1500 1500 1500 shRNAs express BRM (Fig. 2 ), whereas -mutant/deficient sh2025 sh5537 ns CTL ns ns lung cancer cell lines (SBC-5 and KP4) that have no or barely 1000 1000 1000 -Dox detectable expression of BRM (Fig. 2B and SI Appendix, Fig. S3) 500 500 500 +Dox (mm³) A

Tumor Tumor did not respond to BRM shRNAs (Fig. 2 and Dataset S3). More Volume Volume NCI-H460 11 17 23 11 17 23 11 17 23 detailed studies of such cancers and preclinical models that sustain Days Days Days proliferation in the absence of BRM and BRG1 will likely provide CTL sh2025 sh5537 insights into potential mechanisms of resistance and inform F G 100 ns *** *** strategies to prevent and/or combat the emergence of resistance. 80 Our study positions BRM as an attractive therapeutic target in -Dox 60 BRG1-deficient cancers. Although BRM and BRG1 are highly

Ki67 40

nuclei related, they display redundant and distinct roles. Whereas in- % Ki67+

NCI-H1299 20 activation of BRG1 is embryonic lethal (27), that of BRM results +Dox in viable animals without any overt deficiencies (28), pointing Dox +- --+ CTL sh2025 sh5537 toward the potential for a good therapeutic window with BRM selective inhibitors. BRM contains a bromodomain and an Fig. 6. BRM knockdown inhibits the growth of BRG1-mutant tumors in ATPase domain, thus presenting multiple attractive avenues vivo. NCI-H1299 cancer cells stably expressing dox-inducible CTL shRNA or for the development of targeted small molecule inhibitors. The two distinct BRM-targeting shRNAs (sh2025 or sh5537) were inoculated into clinical importance of these findings is highlighted by the prev- mice. Tumor-bearing mice were treated for with either vehicle or dox. (A) BRG1 Western blot of tumor BRM and VINCULIN (loading control) after 7 d of alence of mutations in several cancers, including lung treatment. (B) Representative images of BRM IHC staining after 7 d of adenocarcinomas. Of note, previous studies have demonstrated treatment. (C) Percentage of nuclei positive for BRM after 7 d of treatment. that SNF5-deficient malignant rhabdoid tumors are selectively Graphs represent mean ± SEM (n = 3 per treatment group). (D and E) NCI- sensitive to BRG1 inhibition (35). Intriguingly, SNF5-deficient H1299 (D) or NCI-H460 (E) cancer cells stably expressing dox-inducible CTL, malignant rhabdoid tumors lack expression of BRM (35), there- sh2025, or sh5537 BRM shRNA were inoculated into mice. When tumor fore raising the possibility that this synthetic lethality may, in fact, 3 volume reached 100–300 mm , mice were treated continuously with either be explained by the codependency of BRG1 on BRM. Further- vehicle diet (black circles) or dox-supplemented diet (white circles). The tu- more, during preparation of this manuscript, another group in- 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 relative dependently reported similar findings in their study of BRG1/ to vehicle-treated group. (F) Representative images of Ki67 IHC staining of BRM synthetic lethality in nonsmall cell lung cancers (36). Our NCI-H1299 tumors after 7-d treatment. (G) Percentage of nuclei positive for discovery of BRM as the top hit from a systematic unbiased Ki67 in NCI-H1299 tumors after 7 d of treatment. Graphs represent mean ± screening approach reinforces the robustness of the BRM/BRG1 SEM (n = 3pertreatmentgroup). synthetic lethal relationship. Based on the results presented in this

3132 | www.pnas.org/cgi/doi/10.1073/pnas.1316793111 Hoffman et al. Downloaded by guest on September 27, 2021 study, we propose a model in which mSWI/SNF mutations lead to Extraction kit (Thermo) by following manufacturer’s recommendations. a hypomorphic complex that promotes tumorigenesis but cannot Immunoprecipitaiton was performed with an anti-BRM antibody (Abcam) tolerate complete inactivation. In this setting, BRG1 mutations and associated SWI/SNF subunits detected by Western blot using standard create a cancer-specific vulnerability that can be therapeutically protocols. Chemiluminescent signal was detected by using SuperSignal West exploited by selectively targeting the residual BRM containing Femto Maximum Sensitivity Substrateor Li-Cor Odyssey. Additional details complex (SI Appendix,Fig.S18). More generally, this model on methods and antibodies used for immunprecipitation and Western predicts that targeting of redundant activities (paralogs) of mu- blotting are provided in SI Appendix, Methods. tated mSWI/SNF subunits may present a broader strategy for blocking the growth of mSWI/SNF-mutated cancers. The find- Functional Characterization of BRM Knockdown Using Inducible shRNA Constructs. ings in this study therefore support a general approach for shRNA sequences targeting BRM cloned into the pLKO-Tet-On inducible vector system. The sequences of the oligonucleotides used and details on the therapeutic intervention for the large collections of mSWI/ cloning and lentiviral production are provided in SI Appendix, Methods. Lung SNF-mutated cancers through targeting of the residual mSWI/ cancer cell lines were infected with lentiviruses carrying BRM shRNAs, and SNF complex. the effect of BRM knockdown in growth and focus formation assays was Materials and Methods determined in the presence or absence of dox-induced shRNA expression. Details for the cell cycle, senescence, and immunofluorescence assays are Library Design, Construction, and Screening. A custom 6,500 element shRNA provided in SI Appendix, Methods. library focused on enzymes involved in epigenetic regulation was constructed by using chip-based oligonucleotide synthesis and cloned as a pool into the In Vivo Efficacy Studies. All animal studies were carried out according to the the pRSI9 lentiviral plasmid (Cellecta). Viral packaging was carried out Novartis Guide for the Care and Use of Laboratory Animals. Mice were in- according to the manufacturers recommended protocol. Each cell line was screened in duplicate, maintained an average minimal representation of oculated s.c. with NCI-H1299 or NCI-H460 cancer cells stably expressing dox- 1,000 cells per sRNA, and harvested after five-population doublings. The inducible CTL nontargeting shRNA or two distinct BRM-targeting shRNAs, representation of each barcode in the library was measured by next gen- and tumor volume was measured twice weekly. At termination of each eration sequencing on an Illumina GA2X. Detailed protocols for the viral study, tumor tissue was collected from each group and processed for im- packaging, transduction, screening, and data analysis are provided in SI munohistochemistry by using standard methods. A detailed description of Appendix, Methods. these xenograft and the immunohistochemistry (IHC) studies is provided in SI Appendix, Methods. Cell Culture, Immunoprecipitation, and Western blotting. NCI-H1299, NCI-H460 NCI-H838, and A549 cells were cultured in recommended media. Nuclear ACKNOWLEDGMENTS. We thank Bill Forrester and Nathan Ross for discussions lysates were prepared by using the NE-PER Nuclear and Cytoplasmic and a critical reading of the manuscript.

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