A Synthetic Lethality–Based Strategy to Treat Cancers Harboring a Genetic Deﬁciency in the Chromatin Remodeling Factor BRG1

Published OnlineFirst July 19, 2013; DOI: 10.1158/0008-5472.CAN-12-4593

Cancer Therapeutics, Targets, and Chemical Biology Research

Takahiro Oike1,2,6, Hideaki Ogiwara1, Yuichi Tominaga3, Kentaro Ito3, Osamu Ando3, Koji Tsuta4, Tatsuji Mizukami1,6, Yoko Shimada1, Hisanori Isomura1,2, Mayumi Komachi6, Koh Furuta4, Shun-Ichi Watanabe5, Takashi Nakano6, Jun Yokota2, and Takashi Kohno1

Abstract The occurrence of inactivating mutations in SWI/SNF chromatin-remodeling genes in common cancers has attracted a great deal of interest. However, mechanistic strategies to target tumor cells carrying such mutations are yet to be developed. This study proposes a synthetic-lethality therapy for treating cancers deficient in the SWI/ SNF catalytic (ATPase) subunit, BRG1/SMARCA4. The strategy relies upon inhibition of BRM/SMARCA2, another catalytic SWI/SNF subunit with a BRG1-related activity. Immunohistochemical analysis of a cohort of non–small- cell lung carcinomas (NSCLC) indicated that 15.5% (16 of 103) of the cohort, corresponding to preferentially undifferentiated tumors, was deficient in BRG1 expression. All BRG1-deficient cases were negative for alterations in known therapeutic target genes, for example, EGFR and DDR2 gene mutations, ALK gene fusions, or FGFR1 gene amplifications. RNA interference (RNAi)–mediated silencing of BRM suppressed the growth of BRG1-deficient cancer cells relative to BRG1-proficient cancer cells, inducing senescence via activation of p21/CDKN1A. This growth suppression was reversed by transduction of wild-type but not ATPase-deficient BRG1. In support of these in vitro results, a conditional RNAi study conducted in vivo revealed that BRM depletion suppressed the growth of BRG1-deficient tumor xenografts. Our results offer a rationale to develop BRM-ATPase inhibitors as a strategy to treat BRG1/SMARCA4–deficient cancers, including NSCLCs that lack mutations in presently known therapeutic target genes. Cancer Res; 1–11. 2013 AACR.

Introduction ma (NSCLC; refs. 5, 6). Meanwhile, inactivating somatic muta- Tyrosine kinase inhibitors are effective against solid tumors tions of genes that encode subunits of the SWI/SNF chromatin- with activating mutations in tyrosine kinase genes, for exam- remodeling complex, such as BRG1/SMARCA4, PBRM1/ ple, the EGFR mutations and ALK fusions harbored by lung BAF180, ARID1A/BAF250A, and ARID2/BAF200, have attracted fi fi adenocarcinomas (1). We and others recently identified RET much interest as they were rst identi ed by genome-wide oncogene fusions in lung adenocarcinoma (2–4), supporting sequencing analyses of cancer cells (7). Such mutations are the importance of tyrosine kinase genes as therapeutic targets. thought to interfere with the functions of the SWI/SNF com- Two other tyrosine kinase gene alterations, the FGFR1 ampli- plex in transcription (7) and DNA double-strand break repair fication and the DDR2 mutation, have been identified as (8), which are likely to be critical for cancer development and/ therapeutic targets in squamous cell carcinoma, which corre- or progression. However, no therapeutic strategies have yet fi sponds to another major type of non–small-cell lung carcino- been developed for speci cally targeting cancer cells harboring these inactivating SWI/SNF mutations. Synthetic-lethality therapy holds great promise. The BRCA1 and BRCA2 genes have a synthetic-lethal relationship with the fi 1 2 Authors' Af liations: Divisions of Genome Biology and Multistep PARP1 gene; the growth of BRCA1- or BRCA2-deficient cancer Carcinogenesis, National Cancer Center Research Institute; 3Oncology Research Laboratories, Daiichi Sankyo Co., Ltd.; Divisions of 4Pathology cells is dependent on PARP1 function. These findings have and Clinical Laboratories and 5Thoracic Surgery, National Cancer Center been translated to the clinic through the development of PARP Hospital, Tokyo; and 6Department of Radiation Oncology, Gunma Univer- – fi sity Graduate School of Medicine, Gunma, Japan inhibitors to treat BRCA1/BRCA2 de cient tumors (9). Syn- thetic-lethality therapy has also been proposed for treatment Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). of cancers de cient in genes involved in DNA mismatch repair and cell metabolism (10–12). Here, we developed a novel Corresponding Author: Takashi Kohno, Division of Genome Biology, fi National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo synthetic-lethality strategy for killing tumor cells de cient in 104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-3542-0807; E-mail: the SWI/SNF subunit BRG1 (also known as SMARCA4). Indeed, [email protected] inactivating somatic BRG1 mutations are present in several doi: 10.1158/0008-5472.CAN-12-4593 cancers, as reported by our group and others (13–15). Many 2013 American Association for Cancer Research. lung cancers (10%–30%) exhibit reduced or absent expression

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of the BRG1 protein due to somatic truncating mutations in the RNA samples are available) were examined by RT-PCR (2, 4). BRG1 gene or unspecified epigenetic alterations (13, 15–18). FGFR1 amplification was examined by conducting FISH analysis Furthermore, BRG1 was recently identified as one of the most (28) on 69 NSCLC samples that were available for analysis frequently mutated genes in medulloblastoma (19, 20) and (Supplementary Materials and Methods). Burkitt lymphoma (21). Each SWI/SNF complex contains either BRG1 or BRM Cell lines (also known as SMARCA2) as its catalytic (ATPase) subunit A427, A549, H157, H460, H520, H522, H661, H1299, H1703, (7, 22). BRG1 and BRM play complementary roles, as indi- H1819, LK2, HCC515, II-18 (NSCLC), U2OS (osteosarcoma), cated by the observation that Brm-deficient mice develop HT1080 (fibrosarcoma), HeLa (cervical cancer), WiDr, HCT116 normally but exhibit upregulated BRG1 expression in somat- (colorectal cancer), HFL-1, and MRC-5 (fibroblast) cells were ic cells (23). Brg1-null heterozygous mice spontaneously cultured in RPMI-1640 (Gibco) supplemented with 10% FBS. develop mammary tumors, and specific alleles of BRG1 are 293FT cells were cultured in Dulbecco's Modified Eagle Medi- associated with cancer predisposition in humans (24). um (DMEM) supplemented with 10% FBS. A549, H1819, H661, Therefore, BRG1/Brg1 acts as a tumor-suppressor gene H157, H1299, A427, H522, and H1703 are lung cancer cell lines (25). The complementary roles of BRG1 and BRM, coupled harboring truncating BRG1 mutations, whereas H520, HeLa, with the involvement of the SWI/SNF complex in multiple LK2, H460, U2OS, and HT1080 carry wild-type BRG1 (13, 17). critical cellular functions, suggest that BRG1 and BRM might The BRG1 mutation status of all the cell lines used in this study have a synthetic-lethal relationship; therefore, we examined is given in Supplementary Table S1. Alterations in BRG1 and the ability of BRM inhibition to specifically kill BRG1-defi- other cancer-related genes were verified in the cell lines we cient tumor cells. In addition, to address the potential examined; some of the results were previously reported (Sup- significance of BRM-inhibitory therapies, we examined the plementary Table S2; ref. 17). characteristics of BRG1-deficient NSCLCs, including the Mutations in BRG1-andBRM-coding regions in HCC515 mutually exclusive relationship between BRG1 deficiencies and II-18 cells, which do not express BRG1, and 16 primary and mutations in therapeutic target genes (EGFR and DDR2 tumors negative for BRG staining were examined by targeted mutations, ALK fusions, and FGFR1 amplifications; Fig. 1A). genome capture and massively parallel sequencing of genomic DNA using an Ion PGM sequencing system and the Ion Materials and Methods TargetSeq Custom Enrichment Kit (Life Technologies). NSCLC cohort Mutations detected were verified by Sanger sequencing of Surgical specimens from 103 patients with NSCLC (51 genomic PCR products. The effect of single-nucleotide var- adenocarcinomas and 53 squamous cell carcinomas), who iants resulting in amino acid changes in the BRG1 ATPase received surgical resection at the National Cancer Center domain, expressed in HCT116 (L1163P) and WiDr (D1284N) Hospital (Tokyo, Japan), between 1997 and 2007, were analyzed colorectal cancer cells (16), was estimated using the Poly- (Table 1 and Supplementary Materials and Methods). The Phen and PolyPhen-2 programs. The substitutions in study was approved by the Institutional Review Board of the HCT116cellsarethoughttoabrogate ATPase activity, but National Cancer Center (Tokyo, Japan). those in WiDr cells are benign (Supplementary Table S3).

Immunohistochemical analysis of BRG1 and BRM siRNA Immunohistochemical analysis was conducted on tissue ON-TARGET plus SMARTpool siRNA (Dharmacon) was microarray sections obtained from anonymized NSCLC sam- used in this study. To knockdown the BRM protein, cells ples. The methods used for immunohistochemical staining and were transfected with siBRM (cat. no: 6597: SMARCA4) using the criteria for BRG1 and BRM positivity are described in the Lipofectamine RNAiMAX (Invitrogen). Nontargeting siRNA Supplementary Materials and Methods. Specific staining for (L-001810-10) was used as a negative control. BRG1 and BRM was confirmed by immunoblot analysis of cancer cell lines (Supplementary Fig. S1). Establishment of cells expressing doxycycline-inducible short hairpin BRM Examination of driver gene mutations To achieve doxycycline-inducible knockdown of BRM, a Genomic DNA obtained from tumor tissues was analyzed for lentiviral vector–based short hairpin RNA (shRNA)–mediated somatic mutations in the EGFR and KRAS genes using the high- conditional gene-expression system [established by Wizner- resolution melting method (26). Mutations in all DDR2 coding owicz and Trono (29)] was used (Supplementary Materials and regions in squamous cell lung carcinoma samples were exam- Methods). H1299 cells carrying a shRNA targeting BRM (H1299- ined by targeted genome capture and massively parallel shBRM) or shRNA targeting the GFP gene (H1299-shControl) sequencing using an Ion PGM sequencing system (Life Tech- were established. HeLa-shBRM and HeLa-shControl cells were nologies) and the HaloPlex Target Enrichment System (Agilent established in the same manner. Technologies). EML4-ALK and KIF5B-ALK fusions were screened by immunohistochemistry using an anti-ALK antibody Immunoblot analysis (Abcam; 5A4), followed by confirmation via reverse-transcrip- Immunoblotting was conducted as previously described (8). tase PCR (RT-PCR) and/or fluorescence/chromogenic in situ The antibodies used in the present study are listed in the hybridization (27). RET and ROS1 fusions (in cases for which Supplementary Materials and Methods.

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A C BRG1(–)/BRM(+) BRG1 expression and other driver BRG1 BRM mutaons in surgical specimens

Synthec-lethal relaonship between BRG1 and BRM in vitro

Growth of BRG(–) tumor by BRM depleon in vivo BRG1(+)/BRM(+) (%) B 9.7 BRG1 BRM 5.8 BRG1–/BRM+ 33.0 51.5 BRG1–/BRM– BRG1+/BRM– BRG1+/BRM+

D Cases → BRG1 Positive Negative BRM Positive Negative EGFR Mutant wt KRAS Mutant wt ALK Fusion wt FGFR1 Amp wt Diff. Poor Mod Well Age ≥67 <67 Sex Male Female Smoking Ever Never Histol. Ad Sq pStage III-IV II I

Figure 1. BRG1 and BRM expression in surgically resected NSCLCs. A, strategy. B, proportion of specimens expressing the BRG1 and BRM proteins. The staining intensity of BRG1 and BRM antibodies in the tumor cell nuclei in each lung cancer sample was scored according to Fukuoka and colleagues (46): 0, no signal; 1, weak; 2, moderate; and 3, strong; comparable with that in salivary gland cells used as the positive control; in addition, the intratumoral fractions of regions with each score (0–3) were determined. The total score for each lung cancer tissue (the sum of the intensity scores and the fractions) was compared with that of noncancerous lung tissues (n ¼ 6). A tumor sample was judged positive (þ) or negative/weak ()when the total score was higher or lower, respectively, than the average total score of the normal lung epithelial tissues. C, representative images of immunohistochemical staining. Top, a BRG1-negative/BRM-positive specimen. Bottom, a BRG1/BRM–positive specimen. The stromal cells in both specimens were positive for BRG1 and BRM. D, BRG1/BRM protein expression and other clinicopathologic factors. Patients are characterized according to BRG1 and BRM staining and driver gene mutations. BRG1, a case of adenocarcinoma with a nonsense BRG1 mutation is dotted. FGFR1, cases not examined for FGFR1 ampliﬁcation are dotted. DDR2 mutations were not detected in all cases of squamous cell carcinoma. wt, wild-type.

Cell survival assay Complementation assay The effect of BRM knockdown on the survival of BRG1- The complementary role of BRG1 was examined in BRM- deficient (undetectable by immunoblot analysis) and BRG1- depleted cancer cells by transfecting cDNAs encoding wild- proficient (detectable by immunoblot analysis) cancer cells type and mutant BRG1 (BRG1K785A, which disrupts the ATP- was evaluated using clonogenic survival assays. Because non- binding pocket in BRG1; ref. 30) into BRG1-null H1299 cells cancerous fibroblasts do not form colonies on culture plates, (Supplementary Materials and Methods). the viability of these cells was determined by examining cellular ATP levels using the CellTiter-Glo Luminescent Cell Cell-cycle analysis Viability Assay Kit (Promega). The detailed procedures for Cells were trypsinized, centrifuged, washed in PBS, and fixed these assays were described in the Supplementary Materials in ice-cold 70% ethanol. The cells were then centrifuged and Methods. again, incubated with PBS containing 200 mg/mL RNase A and

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Table 1. Patient characteristics and their association with lost/reduced BRG1/SMARCA4 expression

P All BRG1 lost/reduced BRG1 retained Variant N (%) N (%) N (%) Univariatea Multivariateb Total patients 103 (100.0) 16 (100.0) 87 (100.0) NT NT Age Median [range] SD 67 [44–82] 8 60 [48–78] 8 67 [44–82] 8 0.094 0.13 >Median 52 (50.5) 5 (31.3) 47 (54.0) Gender Male 73 (70.9) 16 (100.0) 57 (65.5) 0.0053 NT Female 30 (29.1) 0 (0.0) 30 (34.5) Histologic cell type Adenocarcinoma 50 (48.5) 8 (50.0) 42 (48.3) 0.90 NT Squamous cell carcinoma 53 (51.5) 8 (50.0) 45 (51.7) Differentiation Well differentiated 38 (36.9) 2 (12.5) 36 (41.4) 0.0011 0.022 Moderately differentiated 34 (33.0) 3 (18.8) 31 (35.6) Poorly differentiated 31 (30.1) 11 (68.8) 20 (23.0) Pathologic stage I 42 (40.8) 5 (31.3) 37 (42.5) 0.078 0.20 II 28 (27.2) 8 (50.0) 20 (23.0) III and IV 33 (32.0) 3 (18.8) 30 (34.5) Smoking habit Never-smoker 26 (25.2) 1 (6.3) 25 (28.7) 0.057 0.17 Ever-smoker 77 (74.8) 15 (93.8) 62 (71.3) EGFR mutation Mutant 27 (26.2) 0 (0.0) 27 (31.0) 0.0095 NT Wild-type 76 (73.8) 16 (100.0) 60 (69.0) KRAS mutation Mutant 10 (9.7) 0 (0.0) 10 (11.5) 0.15 NT Wild-type 93 (90.3) 16 (100.0) 77 (88.5) ALK fusion Positive 1 (1.0) 0 (0.0) 1 (1.1) 0.67 NT Negative 102 (99.0) 16 (100.0) 86 (98.9) FGFR1 ampliﬁcation Positive 1 (1.0) 0 (0.0) 1 (1.1) 0.67 NT Negative 68 (99.0) 12 (100.0) 56 (98.9) BRM expression Lost or reduced 40 (38.8) 6 (37.5) 34 (39.1) 0.91 NT Retained 63 (61.2) 10 (62.5) 53 (60.9)

Abbreviation: NT, not tested. aAssociation was examined by Fisher exact test. bAssociation was examined by logistic regression test. Variables showing associations with P < 0.1 in the univariate analysis were subjected to this analysis. Gender and EGFR mutation were not included; they are not suitable for this analysis because no females or EGFR mutants were present in the "BRG lost/reduced" group.

5 mg/mL propidium iodide, and analyzed for cell-cycle distri- Fluorescence microscopy bution by Guava ﬂow cytometry (Millipore). Cells were stained with 40,6-diamidino-2-phenylindole (DAPI) and examined for senescence-associated heterochro- Senescence-associated b-galactosidase staining matic foci (SAHF) and signs of mitotic catastrophe (Supple- Senescence-associated b-galactosidase (SA-b-gal) staining mentary Materials and Methods). was conducted using the Senescence b-Galactosidase Staining Kit (Cell Signaling Technology). The percentage of SA-b-gal– Long-term in vitro cell proliferation assay positive cells was determined from three different ﬁelds per H1299-shBRM and HeLa-shBRM cells were seeded in 60-mm sample. dishes (1 105 cells/dish) in media with or without 0.1 mg/mL

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doxycycline, and then cultured. The cells were trypsinized and DDR2 mutations (Fig. 1D; Table 1; Supplementary Fig. S3; counted every 3 days, and after each count, 1 105 cells were refs. 5, 6). These findings suggest that most BRG1-deficient reseeded in new dishes with fresh medium. Accumulated cell NSCLCs are not suitable for current molecularly targeted numbers were calculated at each passage, based on the total therapies based on tyrosine kinase inhibitors (1). Of the 16 number of cells and the dilution ratio at the time of passage. BRG1-deficient cases, 10 (62.5%, 9.7% of all cases studied) The experiment was carried out in duplicate. expressed BRM.

Mouse xenograft model BRM-dependent growth of BRG1-deficient cancer cells The effect of BRM ablation on the growth of H1299-shBRM We then compared the effect of siRNA-mediated BRM and H1299-shControl tumor xenografts was examined in ablation on in vitro growth of BRG1-defi(ncient ¼ 10) and BALB/c-nu/nu mice (Supplementary Materials and Methods). BRG1-proficient (n ¼ 8) cancer cell lines, using a clonogenic All experiments were approved by the Ethical Committee on survival assay (Fig. 2A). Most (8 of 10) BRG1-deficient cells carry Animal Experiments at the National Cancer Center. truncating BRG1 mutations/deletions (Supplementary Table S2; refs. 13, 17). Of the remaining two BRG1-deficient cell lines, Statistical analysis we detected a homozygous nonsense mutation in one Logistic regression analysis was conducted using JMP (ver (HCC515) by targeted genome capture sequencing (Supple- 5.1). Fisher exact test and Student t test were conducted using mentary Fig. S2), but no BRG1 mutation in the other (II-18). On StatMate III (ver 3.17). P < 0.05 was considered statistically the other hand, BRG1-proficient cancer cells did not harbor significant. Experiments were carried out in triplicate unless such deleterious BRG1 mutations (except HCT116 cells, which otherwise stated. harbor a heterozygous missense mutation that is likely to disrupt the BRG1 ATPase domain; ref. 15). Results We observed suppression of colony formation in all BRG1- Characteristics of BRG1-deficient NSCLCs deficient cells tested, but not in BRG1-proficient lines other We first examined the characteristics of BRG1-deficient than HCT116 (Fig. 2B and C). The mean values of the surviving NSCLCs. Because BRG1 has been reported to play a role in fraction of cancer cell lines are summarized according to the cell differentiation (31), the examined cases were selected to BRG1 and BRM expression status in Fig. 2D. Survival of BRG1- include similar numbers of adenocarcinomas and squamous deficient cells upon BRM knockdown was significantly lower cell carcinomas of the three differentiation grades (well, mod- than that of BRG1-proficient lines (P ¼ 6.3 10 6 by Student t erately, and poorly differentiated; Table 1). This approach test). The BRM protein was undetectable, or detectable only at allowed an efficient analysis of the association between BRG1 trace levels, in three BRG1-deficient cell lines, A427, H522, and expression and tumor differentiation. Immunohistochemical H1703 (Fig. 2A and Supplementary Fig. S4A). These findings are analysis revealed that BRG1 protein expression was either low consistent with a report showing that a fraction of lung cancer or absent in 16 of 103 (15.5%) NSCLCs (Fig. 1B and C). Low or cells lack, or express at low levels, both BRG1 and BRM (Fig. 1B; absent BRG1 expression was more prevalent in poorly differ- ref. 14). Conceivably, the effect of siRNA-mediated BRM knock- entiated tumors (Table 1 and Fig. 1D), and was also prevalent in down in these three cell lines was less evident than in the other males and smokers. Mutational analysis of BRG1 and BRM in 16 seven. Thus, among BRG1-deficient cell lines, the suppression cases negative for BRG1 staining revealed that 1 case had of colony formation by BRM knockdown was more prominent nonsense BRG1 mutations (Supplementary Fig. S2), whereas in cell lines that originally expressed the BRM protein (P ¼ 4.5 the remaining 15 did not, implying that both genetic altera- 10 9 by Student t test; Fig. 2D). tions and epigenetic silencing of the BRG1 gene can cause BRM knockdown did not affect the growth of the noncan- BRG1 deficiency, as previously reported (18). On the other cerous fibroblast cell lines HFL-1 and MRC-5, which express hand, deleterious BRM mutations were not detected in these 16 both BRG1 and BRM (Fig. 3A and B). Wild-type BRG1 cDNA cases including 5 that were negative for BRM staining. complemented the sensitivity of BRG1-deficient H1299 cells to BRM siRNA-mediated growth inhibition (Fig. 3C and D), Driver gene mutations in BRG1-deficient NSCLCs whereas cDNA of an ATPase mutant with a disrupted ATP- Next, we analyzed representative therapeutically relevant binding pocket, BRG1K785A (30), did not (BRG1K785A is indicat- gene mutations (EGFR and KRAS gene mutations and ALK ed as KA in Fig. 3C and D). These results indicated that BRG1- fusions) in the 103 NSCLC cases. The 16 BRG1-deficient cases deficient cancer cells depend on BRM for growth, and that this were negative for mutations in all three driver genes, including phenotype is caused by a lack of BRG1 ATPase activity. the two therapeutic targets (EGFR mutation and ALK fusion; Fig. 1D, Table 1). We also analyzed other driver gene BRM depletion induces senescence in BRG1-deficient mutations, RET and ROS1 fusions (2–4), in the 9 BRG1-deficient cancer cells cases for which tumor tissue RNA was available, but we To examine the long-term effects of BRM depletion, we detected no fusions. Recent whole-genome analyses have also prepared H1299 (BRG1-deficient) and HeLa (BRG1-proficient) suggested that BRG1 mutations are prevalent in tumors with- cells expressing either a doxycycline-inducible shRNA target- out EGFR mutations or ALK fusions (Supplementary Table S4; ing BRM (H1299-shBRM and HeLa-shBRM; refs. 29, 34) or a refs. 32, 33). In addition, all the BRG1-deficient cases were also nontargeting shRNA (H1299-shControl and HeLa-shControl). negative for two other alterations, FGFR1 amplifications and To confirm the effects of BRM knockdown, we used a different

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A 9 C H1703 H1819 HT1080 H129 HCC515 A427 H522 II-18 A549 H661 H157 U2OS H520 HeLa H460 LK2 WiDr HeLa HCT116 (kD) H661 HCC515 II-18 A549 H1819 H157 A427 H522 −250 H1299 BRG1 siBRM – + – + – + – + – + – + – + – + – + −150 BRM −100 1.00 0.24 1.000.14 1.00 0.07 1.00 0.21 1.00 0.07 1.00 0.22 1.00 0.05--- - − 75 α-Tub − 50 − 37 − 25 H520 U2OS HT1080 HeLa LK2 WiDr H460 H1703 − 20 HCT116 siBRM − 15 – + – + – + – + – + – + – + – + – + − 10 BRM

BRM – – 1.00 0.17 1.000.08 1.00 0.02 1.00 0.10 1.00 0.27 1.00 0.20 1.00 0.10 1.00 0.03 α -Tub α-Tub

B D 1.2 P = 4.5×10–9

1 P = 6.3×10–6 1.2 ᧦ 0.8 ᧦

᧦ 1

0.6 ᧦ 0.8 ᧦ 0.6 0.4 ᧦

Surviving fraction siControl 0.4 ᧦ ᧦ ᧦ 0.2 ᧦ ᧦ ᧦ siBRM Surviving fraction ᧦ 0.2 ᧦ ᧦ ᧦ ᧦᧦ ᧦ 0 ᧦ 0 BRG1 ––+ LK2 II-18 WiDr H520 H460 A549 H661 A427 H522 H157 HeLa

U2OS ± H1299 H1819 H1703 BRM + + HT1080 HCT116 HCC515 BRG1-deficient BRG1-proficient

Figure 2. Differential sensitivity of BRG1-deficient and -proficient cell lines to BRM knockdown. A, BRG1 and BRM protein expression in human cancer cell lines. Immunoblot analysis of BRG1-deficient (left) and BRG1-proficient (right) cancer cell lines. Blots of whole-cell extracts were probed with antibodies against BRG1, BRM, and a-tubulin (a-Tub; loading control). B, survival of BRG1-deficient and -proficient cancer cells after BRM knockdown. BRG1-deficient (left) and -proficient (right) cells were transfected with BRM-targeting siRNA (siBRM) or nontargeting siRNA (siControl) for 48 hours and then assayed for colony formation. The surviving fraction of siBRM-treated cells at 10 days was calculated as a ratio (number of colonies formed by siBRM-treated cells/number of colonies formed by siControl-treated cells). Data are shown as mean SD. Asterisks show significant differences in surviving fraction between siBRM- and siControl-treated cells, as determined by Student t test (, P < 0.05; , P < 0.001). C, BRM protein expression in cancer cells after siRNA-mediated knockdown. Cells were transfected with siBRM or siControl for 48 hours, harvested, and subjected to immunoblot analysis. Blotsof whole-cell extracts were probed with antibodies against BRM and a-tubulin (loading control). The expression levels of BRM in siBRM-treated cells are shown relative to those in siControl-treated cells. Knockdown efficiency in A427, H522, and H1703 cells could not be assessed because of their low endogenous BRM expression levels. D, surviving fraction of cancer cell lines according to BRG1 and BRM expression status. The mean values of the surviving fractions of each cell line obtained in Fig. 2B are shown. Cell lines are grouped according to deficiency () and proficiency (þ) of BRG1 and BRM protein expression. Significance of differences in the mean values of surviving fraction between BRG1-deficient cell lines and BRG1-proficient cell lines, and BRG1- deficient/BRM-proficient cell lines and BRG1-proficient cell lines were evaluated using Student t test.

BRM target site from the one used in the siRNA experiments. or the number of cells exhibiting distinct nuclear lobation, The results of the siRNA clonogenic assay were confirmed indicating that ablation of BRM does not induce apoptosis or using this system (Supplementary Fig. S5). We next examined mitotic catastrophe (Fig. 4A and C; ref. 37). Senescence is an the mechanism underlying growth inhibition in H1299-shBRM irreversible state of growth arrest that is maintained by p21 cells. Seven days after doxycycline treatment, H1299-shBRM (38); therefore, we examined p21 expression in H1299-shBRM cells, but not HeLa-shBRM cells, exhibited G1 arrest with an cells in which BRM was reexpressed after doxycycline removal enlarged and flattened morphology, suggesting senescence (Fig. 4D). In these cells, p21 expression was maintained at (Fig. 4A and Supplementary Fig. S6). Biomarkers of senescence the level observed in BRM knockdown cells. Consistent with (p21/CDKN1A expression, SA-b-gal staining, andSAHF; refs. 35, this, cells reexpressing BRM exhibited suppressed clonogenic 36) were observed in doxycycline-treated H1299-shBRM cells, growth activity. siRNA-mediated knockdown of BRM caused but not in HeLa-shBRM cells (Fig. 4B and C). Doxycycline significant increases in SA-b-gal positivity and the number of treatment did not affect the proportion of cells in sub-G1-phase cells in G1-phase in three BRG1-deficient NSCLC cell lines

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HFL-1 MRC-5 A B 10 siControl siControl MRC-5 HFL-1 (kD) 8 −250 siBRM siBRM BRG1 −150 6 −100 4 siBRM – + siBRM –+

BRM Cell viability BRM BRM 2 1.00 0.10 1.00 0.07 α-Tub α-Tub α-Tub 0 6420 6420 Day Day C D 40 siControl siBRM siControl/- siControl/BRG1 WT –WTKA–WTKA siControl/BRG1 KA BRM 30 siBRM/- 1.00 – – 0.00 0.00 0.00 siBRM/BRG1 WT BRG1 β siBRM/ BRG1 KA -Actin 20 P < 0.05

Cell viability P = 0.30 10

0 76543210 (day)

Figure 3. Effect of BRM knockdown on cell growth in noncancerous cells and in BRG1-deﬁcient H1299 cells complemented with wild-type BRG1 cDNA. A, BRG1 and BRM protein expression in HFL-1 and MRC-5 ﬁbroblasts. Blots of whole-cell extracts were probed with antibodies against BRG1, BRM, and a-tubulin (a-Tub; loading control). B, viability of HFL-1 and MRC-5 cells subjected to siRNA-mediated knockdown of BRM. Cells transfected with siBRM or siControl were cultured for 48 hours and then subjected to CellTiter-Glo assay (Promega) and immunoblot analysis. Cell viability measured by CellTiter-Glo assay is expressed as a ratio relative to the viability assessed at day 0. Data are shown as mean SD. For immunoblot analyses, blots of whole- cell extracts were probed with antibodies against BRM and a-tubulin (loading control). The expression levels of BRM in siBRM-treated cells are shown relative to those in siControl-treated cells. C and D, BRG1 cDNA complementation assay. Twenty-four hours after transfection with siBRM or siControl, H1299 cells were transfected with a plasmid expressing wild-type (WT) or ATPase-mutant (KA) BRG1 protein. Twenty-four hours later (day 0), the cells were seeded into a 96-well plate. On day 2, the cells were retransfected with siRNAs. Cell viabilities measured on days 0, 1, 4, and 7 are shown in C. P values were calculated using Student t test. Expression of BRM and BRG1 proteins on day 1, determined by immunoblot analysis, is shown in D. b-Actin was used as the loading control. The expression levels of BRM in siBRM-treated cells are shown relative to those in siControl-treated cells without cDNA complementation. Data are shown as mean SD of three replicates per treatment condition.

(H1299, A549, and H157), but not in three BRG1-proficient nu mice were injected subcutaneously with H1299-shBRM NSCLC cell lines (H520, LK2, and H460; Fig 4E–G; Supplemen- or H1299-shControl cells. After 3 weeks, when the injected tary Fig. S7). Taken together, these results suggest that BRM cells had formed tumor nodules measuring approximately knockdown specifically suppresses the growth of BRG1-defi- 100 mm3, the animals were randomly divided into two cient cells by inducing senescence. groups and fed a diet containing doxycycline (200 ppm) or a control diet for 1 month. In doxycycline-fed mice, the BRM-dependent growth of BRG1-deficient cancer cells H1299-shBRM xenografts were significantly smaller than in vivo the H1299-shControl xenografts. The number of BRM-depleted H1299-shBRM cells was approximately 1,000 times lower than that of nondepleted Discussion cells after 4 weeks of culture, whereas the number of HeLa- This study suggests that BRM is a novel therapeutic target shBRM cells did not change (Fig. 5A). This observation for synthetic-lethality therapy for BRG1-deficient cancers. To suggests that inhibition of BRM specifically suppresses the the best of our knowledge, this is the first report proposing a long-term growth of BRG1-deficient cells (i.e., over at least a therapeutic strategy that specifically targets and kills cancer month). To further confirm these data under more phys- cells harboring inactivating mutations in SWI/SNF chromatin- iologically relevant conditions, we introduced H1299- remodeling genes. In the future, the use of this strategy needs to shBRM and H1299-shControl cells into an in vivo condi- be further validated in vivo using multiple lines of BRG1- tional RNA interference (RNAi) model (Fig. 5B). BALB/c-nu/ deficient and -proficient cancer cells.

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A B C D Normal SAHF H1299 -shBRM HeLa -shBRM H1299 -shBRM HeLa -shBRM Control Dox – + – + Dox – + – + 07 12(day) BRM BRM 0.161.00 0.051.00 Dox WB & seed 1.00 0.02 1.00 0.09 p21 BRM KD α-Tub 07 12(day) α-Tub MC Dox Dox off WB & seed ᧦ ᧦ Dox– 100 50 Dox+ BRM

(%) KD→Exp 07 12(day) 80 40 Dox– 25 60 30 Dox+ ᧦ 1.2 20 40 20 1 0.8 20 (%) cells -gal–positive 15 β 10 0.6 Control BRM KD KD → Exp Cell-cycle phase Cell-cycle

SA- 10 0 0 0.4 BRM 0.2 ᧦ ᧦ Sub-G 5 0.151.000.96 1 Dox– 0 G2–M p21 Surviving fraction S (%) cells SAHF/MC–positive 0 SAHF MC α-Tub G1 Dox+ Control BRM KD KD → Exp

EFBRG1-deficient BRG1-deficient BRG1-proficient G BRG1-deficient BRG1-proficient H1299 A549 H157 siBRM –+ –+ –+ H1299 A549 H157 H520 LK2 H460 H1299 A549 H157 H520 LK2 H460 BRM ᧦ ᧦ ᧦ ᧦ 0.11.0 0.131.0 0.081.0 60 ᧦ 100 α-Tub 80 40 BRG1-proficient 60 H520 LK2 H460 ᧦ 40 siBRM –+ –+ –+ 20 BRM 20 -ga–positive cells (%) cells -ga–positive

1.00 0.11 1.000.12 1.00 0.08 β 0 (%) phase Cell-cycle 0 α-Tub SA- siControl siBRM Sub-G1 G2–M S G1

Figure 4. Effect of BRM knockdown on growth inhibition in BRG1-deficient cancer cells. A–D, H1299-shBRM and HeLa-shBRM cells were cultured in the presence or absence of doxycycline (Dox) for 7 days before analysis. H1299 cells carrying a doxycycline-inducible shRNA specific for GFP were used as a negative control (H1299-shControl). A, cell-cycle profile. Top, immunoblot analysis of BRM and a-tubulin (a-Tub). The expression level of BRM in doxycycline-treated cells is shown relative to the level in untreated cells. Bottom, cell-cycle profiles determined by flow cytometry. , a significant difference in the fraction of cells in G1-phase between doxycycline-treated and -untreated cells as determined by Student t test (P < 0.01). B, expression of SA-b-gal and p21. Top, immunoblot analysis of BRM, p21, and a-tubulin. The expression level of BRM in doxycycline-treated cells is shown relative to that in untreated cells. Middle, percentage of SA-b-gal–positive cells. , a significant difference in the percentage of SA-b-gal–positive cells between doxycycline- treated and -untreated cells as determined by Student t test (P < 0.001). Bottom, representative micrographs showing cells stained for SA-b-gal (blue). Scale bars, 100 mm. C, SAHF and mitotic catastrophe (MC) as revealed by nuclear DAPI staining. Top, representative images showing normal nuclear (Normal), SAHF, and mitotic catastrophe patterns in doxycycline-treated H1299-shBRM cells. Scale bars, 10 mm. Bottom, percentage of cells exhibiting SAHF and mitotic catastrophe. , a significant difference in the percentage of SAHF-positive cells between doxycycline-treated and -untreated cells as determined by Student t test (P < 0.001). D, p21 expression and clonogenic survival of H1299 cells after BRM knockdown followed by reexpression of BRM. A schematic of the experimental protocol is shown at the top. H1299-shBRM cells were cultured in the presence of doxycycline for 7 days (BRM KD) and then cultured in the absence of doxycycline for an additional 5 days (KD!Exp). The cells were then harvested and subjected to immunoblot analysis (WB). BRM KD and KD!Exp cells were seeded and cultured for an additional 10 days in the presence or absence of doxycycline, respectively, to allow colony formation. H1299-shBRM cells cultured in the absence of doxycycline were used as a control. Bottom left, immunoblot analysis of BRM, p21, and a-tubulin. The expression levels of BRM in BRM KD cells and KD!Exp cells are shown relative to the level in control cells. Bottom right, clonogenic survival assay. The surviving fraction of BRM KD and KD!EXP cells is expressed as a ratio (number of colonies formed by BRM KD and KD!EXP cells/number of colonies formed by control cells). Data are shown as mean SD. , significant decreases in the surviving fraction relative to that of control cells as determined by Student t test (P < 0.001). E–G, BRG1-deficient (H1299, A549, and H157) and BRG1-proficient (H520, LK2, and H460) NSCLC cells were transfected twice (days 1 and 3) with BRM-targeting siRNA (siBRM) or nontargeting siRNA (siControl). On day 7, the cells were subjected to subsequent analyses. E, immunoblot analysis of BRM and a-tubulin (loading control). The expression levels of BRM in siBRM-treated cells are shown relative to those in siControl-treated cells. F, positivity for SA-b-gal staining. , significant differences in the percentage of SA-b-gal–positive cells between siBRM-treated and siControl-treated cells as determined by Student t test (P < 0.005). G, cell-cycle profiles determined by flow cytometry. , significant differences in the fraction of cells in G1-phase between siBRM-treated and siControl-treated cells, as determined by Student t test (P < 0.005).

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Figure 5. Suppression of BRG1- ﬁ de cient cancer cell growth by BRM A H1299-shBRM B H1299-shBRM knockdown both in vitro and in vivo. 15 1,500 A, long-term proliferation of H1299 Dox– Dox– ) and HeLa cells after BRM 3 Dox+ ) 13 Dox+ knockdown. H1299-shBRM and 10 HeLa-shBRM cells were cultured in (mm 1,000 ᧦ the presence or absence of 11 doxycycline (Dox) for 4 weeks. The cells were trypsinized and counted, 9 volume and 1 105 cells were reseeded into 500 60-mm dishes and given fresh medium every 3 days. Accumulated 7 cell numbers were calculated on the Cell number (log basis of the cell number at each 5 0 passage. B, growth of H1299 28211470 (day) 3020100 (day) xenografts in mice. H1299-shBRM and H1299-shControl cells were HeLa-shBRM H1299-shControl implanted subcutaneously into 15 Dox– BALB/c-nu/nu mice. After 3 weeks Dox– ) Tumor 4,000 ) 3

(when the tumors reached 10 13 Dox+ Dox+ 100 mm3), the mice were randomly divided into two groups and either 3,000 fed a diet containing doxycycline 11 (Doxþ) or a control diet (Dox). 2,000 Tumors were measured twice per 9 week. Data represent the mean ﬁ 1,000 SD. ,signi cant differences in the Cell number (log 7 tumor volume between the (mm volume Tumor doxycycline-fed mice and the 0 controls, as determined by 5 100 20 30 Student t test (P < 0.05). 28211470 (day) (day)

In our cohort, all the BRG1-deficient tumors were negative well as in DNA double-strand break repair (7, 8, 31). BRG1 is for EGFR mutations and ALK fusions, both of which can be involved in the expression of genes that regulate cell growth targeted by existing molecular therapies using tyrosine and stem-cell properties. However, a preliminary study indi- kinase inhibitors (1). This negative association of BRG1- cated that the expression of representative genes, including inactivating mutations with therapeutic target mutations those encoding E2F1 and cyclin D1, does not differ significantly is consistent with two recent genome-wide mutation anal- between BRG1-proficient and -deficient cells depleted of BRM. yses of lung adenocarcinoma (Supplementary Table S4; Thus, the mechanisms underlying the synthetic-lethal rela- refs. 32, 33). Indeed, BRG1-deficient NSCLC cell lines are tionship between BRG1 and BRM remain unknown. Of concern resistant to tyrosine kinase inhibitors that target EGFR is the subset of NSCLC cases (5.8%; Fig. 1B) that lack both BRG1 (e.g., A549, H1819, and H1299; ref. 39) or ALK (A549; ref. 40). and BRM expression (14, 22, 42). BRM deficiency is likely to be Furthermore, those BRG1-deficient tumors were also nega- due to epigenetic alterations as previously reported (43). The tive for FGFR1 amplifications and DDR2 mutations, potential existence of these cases suggests that a portion of BRG1- therapeutic targets recently identified in squamous cell lung deficient tumors will not respond well to therapies that target carcinoma (5, 6). These findings highlight the importance of BRM, as suggested by our experiment using cancer cell lines establishingatreatmentstrategy for targeting BRG1-defi- (Fig. 2B). Such tumors may have acquired the ability to cient tumors. BRG1 and BRM proteins have ATPase activ- maintain cell growth through complementation by chroma- ities and function as helicases to alter chromatin structure. tin-remodeling proteins other than BRG1 and BRM, although ATPases are a druggable target (41); therefore, BRM-target- our preliminary analysis did not reveal complementation by ing therapy using specific inhibitors is a promising treat- overexpression of other SWI/SNF and chromatin-remodeling ment of lung cancers that are not amenable to existing proteins (Supplementary Fig. S4B). Studies of the molecular molecularly targeted therapies (1). To the best of our knowl- mechanisms underlying the maintenance of cell growth in the edge, specific pharmacologic inhibitors against ATPases absence of both BRG1 and BRM would be valuable, because it is involved in chromatin remodeling have not been developed; possible that BRG1-deficient cells could acquire resistance to therefore, we are currently seeking to identify substrates that BRM-targeted therapy. inhibit BRM-ATPase activity. The pathogenic significance of inactivating somatic muta- The results of this study show that BRG1 and BRM have a tions in genes encoding the subunits of the SWI/SNF chro- synthetic-lethal relationship. BRG1 functions as a key enzyme matin-remodeling complex in human cancers is largely in SWI/SNF chromatin remodeling during transcription, as unknown (7). This study sheds light on this issue by showing

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that BRG1-deficient tumors are often poorly differentiated. Authors' Contributions A previous report showed that BRG1 promotes cell differen- Conception and design: T. Oike, H. Ogiwara, S.-I. Watanabe, T. Nakano, J. Yokota, T. Kohno tiation, that is, the inactivation of BRG1 enables cancer cells to Development of methodology: T. Oike, H. Ogiwara, O. Ando, S.-I. Watanabe maintain undifferentiated gene-expression programs (31). Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Oike, H. Ogiwara, Y. Tominaga, K. Ito, O. Ando, K. Therefore, loss of BRG1 function may be responsible for tumor Tsuta, T. Mizukami, Y. Shimada, H. Isomura, M. Komachi, K. Furuta, S.-I. dedifferentiation. Our results also indicated that BRG1 defi- Watanabe ciency is prevalent in tumors of males and smokers (Table 1), Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Oike, H. Ogiwara, O. Ando, M. Komachi, S.-I. consistent with recent genome-wide mutational studies (Sup- Watanabe plementary Table S5; refs. 40, 41). Therefore, BRG1 deficiency Writing, review, and/or revision of the manuscript: T. Oike, H. Ogiwara, O. may play a role in lung carcinogenesis preferentially in male Ando, S.-I. Watanabe, T. Nakano, T. Kohno Administrative, technical, or material support (i.e., reporting or orga- and/or smokers, although the underlying mechanisms remain nizing data, constructing databases): H. Ogiwara, O. Ando, S.-I. Watanabe, J. unclear. Yokota Taken together, the results of this study identify BRM as a Study supervision: H. Ogiwara, S.-I. Watanabe, T. Nakano, J.Yokota candidate target molecule for synthetic-lethal therapy for fi Acknowledgments BRG1-de cient lung cancers. Immunohistochemical analy- The authors thank A.F. Gazdar, J.D. Minna, and M. Perucho for providing cell ses revealed that approximately 10% of NSCLCs are BRG1- lines. Cell lines were also obtained from American Type Culture Collection. The deficient and BRM-proficient, and are therefore predicted authors also thank K. Haraguchi and H. Iba at the University of Tokyo (Tokyo, Japan), N. Tsuchiya and K. Shiraishi at the National Cancer Center Research to respond to therapies that target BRM. Inactivation of Institute (Tokyo, Japan) for technical assistance, and our colleagues at the BRG1 also occurs in other cancers, including pancreatic, Department of Radiation Oncology, Gunma University (Gunma, Japan) for their skin, and brain cancers (44). BRG1 was recently identified as sincere encouragement. a gene that is frequently mutated in medulloblastomas Grant Support (subgroup 3; ref. 45), which lacks known driver gene muta- This study was supported in part by Grants-in-Aid from the Ministry of tions (19, 20). Thus, it is possible that therapies targeting Education, Culture, Sports, Science, and Technology of Japan for Scientific BRM will be suitable for treating a variety of cancers. Research on Innovative Areas (22131006), from the Japan Society for the Promotion of Science for Young Scientists (B) KAKENHI (23701110), and from Inactivating mutations in chromatin-remodeling genes oth- the Ministry of Health, Labor and Welfare for the Third-term Comprehensive 10- er than BRG1,suchasPBRM1/BAF180, ARID1A/BAF250A, year Strategy for Cancer Control; and by Management Expenses Grants from the fi Government to the National Cancer Center. The National Cancer Center Biobank and ARID2/BAF200,havebeenidenti ed in several common is supported by the National Cancer Center Research and Development Fund of cancers. Synthetic lethality–based therapeutic strategies Japan. targeting tumors harboring these mutations warrant fur- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in ther study. accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest Received December 16, 2012; revised May 30, 2013; accepted June 17, 2013; No potential conﬂicts of interest were disclosed. published OnlineFirst July 19, 2013.

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A Synthetic Lethality−Based Strategy to Treat Cancers Harboring a Genetic Deficiency in the Chromatin Remodeling Factor BRG1

Takahiro Oike, Hideaki Ogiwara, Yuichi Tominaga, et al.

Cancer Res Published OnlineFirst July 19, 2013.

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