The SMARCA2/4 ATPase Domain Surpasses theBromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue andPFI-3 Inhibitor StudiesBhavatarini Vangamudi1, Thomas A. Paul2, Parantu K. Shah1, Maria Kost-Alimova1,Lisa Nottebaum2, Xi Shi1, Yanai Zhan1, Elisabetta Leo1, Harshad S. Mahadeshwar1,Alexei Protopopov1, Andrew Futreal3,Trang N.Tieu1, Mike Peoples1,Timothy P. Heffernan1,Joseph R. Marszalek1, Carlo Toniatti1, Alessia Petrocchi1, Dominique Verhelle2,Dafydd R. Owen4, Giulio Draetta1, Philip Jones1,Wylie S. Palmer1, Shikhar Sharma2, andJannik N. Andersen1
Abstract
The SWI/SNF multisubunit complex modulates chromatinstructure through the activity of two mutually exclusive cata-lytic subunits, SMARCA2 and SMARCA4, which both contain abromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutanttumors, including SMARCA4-deficient lung cancer; however,the contribution of conserved, druggable protein domains tothis anticancer phenotype is unknown. Here, we functionallydeconstruct the SMARCA2/4 paralog dependence of cancer cellsusing bioinformatics, genetic, and pharmacologic tools. Weevaluate a selective SMARCA2/4 bromodomain inhibitor(PFI-3) and characterize its activity in chromatin-binding andcell-functional assays focusing on cells with altered SWI/SNFcomplex (e.g., lung, synovial sarcoma, leukemia, and rhabdoidtumors). We demonstrate that PFI-3 is a potent, cell-permeableprobe capable of displacing ectopically expressed, GFP-taggedSMARCA2-bromodomain from chromatin, yet contrary to
target knockdown, the inhibitor fails to display an antiproli-ferative phenotype. Mechanistically, the lack of pharmacologicefficacy is reconciled by the failure of bromodomain inhibitionto displace endogenous, full-length SMARCA2 from chromatinas determined by in situ cell extraction, chromatin immuno-precipitation, and target gene expression studies. Furthermore,using inducible RNAi and cDNA complementation (bromodo-main- and ATPase-dead constructs), we unequivocally identifythe ATPase domain, and not the bromodomain of SMARCA2,as the relevant therapeutic target with the catalytic activitysuppressing defined transcriptional programs. Taken together,our complementary genetic and pharmacologic studies exem-plify a general strategy for multidomain protein drug-targetvalidation and in case of SMARCA2/4 highlight the potentialfor drugging the more challenging helicase/ATPase domain todeliver on the promise of synthetic-lethality therapy. Cancer Res;75(18); 3865–78. �2015 AACR.
IntroductionEpigenetic dysregulation plays a fundamental role in the devel-
opment of cancer (1). Large-scale genome sequencing has uncov-
ered recurrent somaticmutations and copy-number (CN) changesin histone-modifying enzymes and chromatin remodeling com-plexes supporting a causal role for altered epigenetic states intumorigenesis (2–4). Although the mechanistic consequencesof these alterations remain poorly understood, it is appreciatedthat such events promote acquisition of cell oncogenic capabil-ities through deregulation of nucleosome-dynamics, gene tran-scription, DNA replication, and repair (5). Indeed, chromatinregulators are emerging as therapeutic targets and inhibitors ofhistone-modifying enzymes, as well as bromodomains, which"read" the histone marks, have recently shown efficacy in pre-clinical and clinical settings through their ability to reverseoncogenic transcriptional programs (6–8).
The Switch/Sucrose Non Fermentable (SWI/SNF) is a multi-subunit chromatin remodeling complex that consists of one oftwo mutually exclusive helicase/ATPase catalytic subunits,SMARCA2 and SMARCA4. Together with core and regulatorysubunits, SMARCA2/4 couple ATP hydrolysis to the perturba-tion of histone-DNA contacts. This sculpting of the nucleoso-mal landscape at promoters provides access to transcription
1Institute for Applied Cancer Science, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Pfizer Oncology ResearchUnit, La Jolla, California. 3Department of Genomic Medicine, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.4Pfizer Worldwide Medicinal Chemistry, Cambridge, Massachusetts.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
T.A. Paul and P.K. Shah contributed equally to this article.
Corresponding Authors: Jannik N. Andersen, XTuit Pharmaceuticals, 700 MainStreet, Cambridge, MA 02139. Phone: 617-990-2235; Fax: 617-863-3677; E-mail:[email protected]; and Shikhar Sharma, Pfizer Oncology Research Unit,10724 Science Center Drive, San Diego, CA 92121. Phone: 858-526-4172; E-mail:[email protected]
factors and cognate DNA elements facilitating both gene acti-vation and repression (9). Because various SWI/SNF subunitsare mutated or lost at high frequency in human tumors(2–4, 10), this complex has garnered considerable attention(11). A tumor-suppressive role has most strongly been dem-onstrated in childhood malignant rhabdoid tumors, in whichthe SMARCB1 (Snf5) subunit is biallelicaly inactivated innearly all cases (10). Accordingly, knockout of mouseSMARCB1 results in fully penetrant and lethal cancers with11 weeks median onset (12). In human synovial carcinoma,recurrent chromosomal translocations, which are diagnostic ofthe malignancy, result in oncogenic fusions (SS18-SSX) thatalter the composition/function of the SWI/SNF complex (13).Pointing to the broader relevance of SWI/SNF in cancers arefrequent inactivating mutations in accessory subunits, includ-ing ARID1A in ovarian and endometrial carcinomas (14, 15),and PBRM1 in renal cell carcinomas (16).
Context-specific molecular vulnerabilities that arise duringtumor evolution represent an attractive class of drug targets;however, the frequency and spectrum of somatic lesions oftenconfound efforts to identify such therapeutic targets solely basedon genomic information (17). To address this challenge, func-tional, unbiased chemical, and genetic loss-of-function (LOF)platforms, which use either drug-like small-molecules or siRNA/shRNA libraries, hold the promise to systematically identifynonobvious target-genotype interactions that might impact clin-ical decisions (17–19). Recently, using genetic LOF approaches,three groups have independently identified SMARCA2 as anessential gene in SMARCA4-deficient lung cancer (20–22) pro-posing a synthetic lethality therapeutic approach. However, itremains unclear whether small-molecule inhibitors of theSMARCA2 bromodomain or ATPase domain can mimic thereported RNAi phenotypes resulting from paralog dependencyin SWI/SNF (11, 23).
Several subunits in the SWI/SNF complex contain bromo-domains, which are evolutionary conserved protein–proteininteraction modules that bind acetyl-lysine on proteins andhistone tails (6, 24). Bromodomains are druggable and fol-lowing the antitumor activity of JQ1 (6), there is interest inbroadly developing small-molecules inhibitors against otherfamily members to dissect their therapeutic potential (1, 6, 24,25). Here, we speculate that SMARCA2/4 bromodomains couldcontribute to either assembly or targeting of the SWI/SNFcomplex to specific genomic loci providing an interventiondrug target rationale. However, because bromodomains arefrequently found in large protein complexes (and often flankedby additional domains involved in chromatin-binding andprotein–protein interactions), RNAi-mediated depletion alonedoes not reveal the contribution of individual domains to theLOF phenotype, representing a specific challenge for drug-targetvalidation.
In this report, we conduct complementary cDNA rescue andpharmacologic studies to explore whether the bromodomainof SMARCA2/4 represents a tractable target in SWI/SNF-mutant cancers. We characterize the PFI-3 bromodomaininhibitor in biochemical assays and across preclinical modelswith altered SWI/SNF complex (lung, synovial sarcoma,leukemia, and rhabdoid tumor cells) and discover that bro-modomain function of SMARCA2/4 is dispensable for tumorcell proliferation, while the catalytic ATPase activity isessential.
Materials and MethodsBioinformatics
Genome sequencing data and CN information were down-loaded from cBioPortal (Supplementary Table S1). Cell linegenomic annotation was from the Sanger (www.cancerrxgene.org) and the Broad Institutes (www.broadinstitute.org/ccle). Out-lier sum statistics (26) and standard software packages forsequence analysis were used.
Cell linesCells obtained from the ATCC were cultured accordingly:
RPMI-1640 (A549, H1299, H157, H520, H460, HeLa, andTHP-1); Iscove's Modified Dulbecco's Medium (MV-4-11);McCoy's 5a Modified Medium (A-204 and G-401) and supple-mentedwith 10%FBS (Gibco). The Aska andYamato cells (OsakaMedical Center) were grown in DMEM (20% FBS). All Cell lineswere mycoplasma negative (LookOut Mycoplasma Kit PCR, Sig-ma) and maintained at low passage (<3 month) after thawingfrom master vials (IACS and Pfizer BioBanks) subjected to shorttandem repeat (STR) profiling of polymorphic loci (PromegaPowerPlex 16 system) with a >80% match criteria for cell lineauthentication.
ImmunoblottingAnalysis was performed on whole-cell lysates (Supplementary
AssaysPFI-3 (PF-06687252) is available from SGC (http://www.
thesgc.org/chemical-probes/PFI-3). Bromodomain selectivitywas measured using ligand binding, site-directed competitiveassays (BROMOscan, DiscoverRx; ref. 27). Cell potency wasmeasured using in situ cell extraction, CellTiter-Glo (Promega)and clonogenic assays (Supplementary Information).
RNAi, plasmids, gene expression, and chromatinimmunoprecipitation
SMARCA2/4 shRNA (SIGMA TRC-collection) and siRNA(ON-TARGET PLUS Dharmacon) sequences are listed in Supple-mentary Table S2. ATPase-dead (K785A) and bromodomainmuta-tions (Y1497F and N1540W) were made in human SMARCA4cDNA (GeneCopoeia #GC-Y3533) using site-directed mutagenesis(QuickChange, Agilent Technologies) with equivalent mutationsin SMARCA2 (GeneCopoeia #GC-Z4424). All cDNAs, subclonedinto lentiviral vectors, were sequence verified and virus genera-tion, infection, and generation of stable cell lines were conductedfollowing standard procedures (Supplementary Information).Gene expression (Affymetrix) data and methods have been depos-ited with NCBI (GSE69088). Chromatin immunoprecipitation(ChIP) and qPCR were conducted as previously described (28).
ResultsGenomic alterations in SWI/SNF across human tumors
To build upon recent meta-analysis (2–4), we first examinedboth SWI/SNFmutation and CN variation drawing on a larger setof patient tumors (n ¼ 10,038) from 45 genome sequencing
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studies (Supplementary Table S1). Clearly, genomic alterations inthe 20 canonical SWI/SNF subunits are highly prevalent (Fig. 1A)occurring in 15%of all cancers (3, 4, 10). Cancers with the highestfrequency of lesions in SWI/SNF subunits are rhabdoid tumors,female cancers, including ovarian, uterine, cervical and endome-trial, lung and gastric adenocarcinoma, melanoma, esophageal,and renal clear cell carcinoma (Fig. 1A and Supplementary TableS1). A tumor-suppressive role of the SWI/SNF complex in thesecontexts has been recognized based on the high frequency ofinactivating mutations, which is further underscored by mousegenetic studies (10, 29). In contrast, SWI/SNF mutations do notemerge as significant recurrent alterations in glioblastoma, thy-roid cancer, multiple myeloma, and acute myeloid leukemia(AML). In AML, SMARCA4 may instead be an oncogene drivingcMYC transcription in concert with BRD4 (7, 11, 30, 31). As such,it appears that cellular and tissue context defines the tumor-suppressive or oncogenic functions of the SWI/SNF complex(5, 11, 23).
SMARCA4 deficiency is prevalent and mutually exclusive toSMARCA2 CN loss in lung cancer
Inprimaryhuman lung adenocarcinoma (LUAD), about half ofthe SMARCA4 mutations are deleterious (nonsense and frame-shift mutations) and occur at a 7% frequency in The CancerGenome Atlas patient samples (Fig. 1B). Overall, SWI/SNF com-plex components are mutated in 71 of 229 patients with anaverage mutation rate of approximately 1.7 per sample. In addi-tion, two copy loss of SMARCA4 is observed in 14 out of 299LUAD cases adding to the fraction of SMARCA4-deficient tumors.Because heterozygous SMARCA4 knockout mice are haploinsuf-ficient and tumor prone (29), we also analyzed copy-numberdriven mRNA expression and conclude that loss of one allele isalso sufficient to decrease SMARCA4 expression (Fig. 1C). Focus-ing on LUAD and lung squamous cell carcinoma (LUSC), map-ping of the genomic annotation onto individual patient samplesrevealed that loss of SMARCA2 and SMARCA4 is largely mutuallyexclusive (Fig. 1D and Supplementary Fig. S1A). Moreover, withrespect to gene expression, outlier statistics identifies SMARCA4,along with ARID1A, as the most significantly altered subunits inthe SWI/SNF complex in LUAD (Fig. 1E) with similar profilesobserved for LUSC (Supplementary Fig. S1B). At the genomelevel, SMARCA4 ranks in the top 5% of all genes with negativeoutlier sum statistics and its bimodal expression profile (Fig. 1F)clearly defines a SMARCA4-deficient patient population.
In good concordance with cell line annotation at the Sangerand the Broad Institutes, SMARCA4 protein expression wasnondetectable by Western blot analysis in approximately20% (12/50) of lung cancer lines (Fig. 1G and SupplementaryFig. S1C–S1E). Of eleven SMARCA4-mutant cell lines, only one(NCI-H2286) displayed measurable protein expression (Sup-plementary Fig. S1F). However, as previously noted, nonde-tectable SMARCA4 protein levels (as assessed by immunoblot-ting) occur more often than predicted from mutation and CNanalysis, suggesting that promoter methylation and epigeneticsilencing may be additional oncogenic mechanisms forSMARCA4 loss (32). Paradoxically, a few cell lines appear tohave nondetectable expression of both SMARCA2 andSMARCA4 (Fig. 1G). However, such complete dual-loss ofSMARCA2/4 is not observed in primary LUAD and LUSCtumors as indicated by both our CN analysis (using bothGISTIC and ABSOLUTE algorithms; Supplementary Fig. S1G)
and gene expression considerations. Although 3% (19/549) ofLUAD patients can be categorized as having low expression ofboth SMARCA2 and SMARC4 (10th percentile; hypergeometricdistribution), the expression profile for SMARCA2 lacks thebimodal distribution characteristic for SMARCA4-null cancers(Fig. 1F). Altogether, the above biomarker assessment (i.e.,SMARCA4 loss) outlines a large, well-defined patient popula-tion in need of novel molecularly targeted therapies.
Genotype-specific vulnerability and paralog dependencyexamined in SMARCA4-reconstituted cells
The recent discovery that SMARCA2 knockdown inhibits thegrowth of SMARCA4-deficient cancer cells (20–22) has opened apotential therapeutic avenue and received considerable attention(11, 23, 33). However, it is unknown whether small-moleculeinhibitors against the ATPase or bromodomain can mimic theRNAi phenotype.Hence, defining the contribution of each domainto the LOF phenotype is required for prioritizing drug discovery toachieve tangible clinical therapeutic endpoints. To explore this, wefirst selected a representative panel of SMARCA4-deficient andproficient lung cancer cells (Fig. 2A) and evaluated multipleSMARCA2 shRNAs for knockdown efficiency (Fig. 2B) and phe-notype. Using either viability (Fig. 2C) or long-term clonogenicassays (Fig. 2D and Supplementary Fig. S2), we observed robust,genotype-specific growth inhibition when SMARCA2was depletedin SMARCA4-deficient cell lines (A549, H1299, and H157). Incontrast, knockdown in SMARCA4-proficient cells (H460, H520,and HeLa) had no effect on viability confirming the reportedsynthetic-lethal SMARCA2/4 interaction (20–22).
Next, we engineered SMARCA4-deficient A549 cells to reex-press a doxycycline-inducible wild-type SMARCA4 cDNA (Fig.2E). Control cells treated with SMARCA2 siRNAs did not grow,whereas the expression of SMARCA4 (þDox) completely restoredgrowth (Fig. 2F and G). Most strikingly, SMARCA4 expressionincreased 5-fold following SMARCA2 depletion (Fig. 2F) indic-ative of compensation, a finding that supports the reciprocalassembly and stability of SMARCA2 and SMARCA4 into theSWI/SNF complex (22).
PFI-3 is a selective, potent, and cell-permeable SMARCA2/4bromodomain inhibitor
To explore pharmacologic inhibition of the SMARCA2 bromo-domain, we next evaluated the small-molecule inhibitor PFI-3(Fig. 3A) discovered through a collaboration between the Struc-tural Genomic Consortium and Pfizer. Biochemically, we deter-mined that PFI-3 binds avidly to both SMARCA2 and SMARCA4bromodomains (BROMOScan Kd's between 55 nmol/L and 110nmol/L) consistent with the binding constant (Kd ¼ 89 nmol/L)measured by isothermal titration calorimetry (www.thesgc.org/chemical-probes/PFI-3). Moreover, using recombinant purifiedbromodomains, we discovered that PFI-3 binds with similaravidity to both the short and long isoform of SMARCA2 revealingthat the alternatively-spliced 18 amino acid insert (34) does notimpair PFI-3 binding (Fig. 3A). Moreover, profiling against 32bromodomains at DiscoveRx (27) confirmed exquisite selectivityversus other subfamilies (Fig. 3C and Supplementary Table S3)expanding the PFI-3 selectivity information obtained using dif-ferential scanning fluorimetry (DSF). In summary, we find thatthere is a good concordance between the ligand competition(BROMOScan) and the direct biophysical binding (DSF) assays
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Figure 1.Genomic analysis of the SWI/SNF complex in human cancer. A, percentage distribution of lesions (mutations and CN changes) in SWI/SNF components acrosstumors profiled by The Cancer Genome Atlas and other laboratories (Supplementary Table S1). B, SWI/SNF mutation spectrum in LUAD (n ¼ 229 tumors; 121mutations). C, correlation of SMARCA4 CN with gene expression (RSEM, RNA-Seq Expression by Expectation Maximization). D, CN loss of SMARCA2/4 is mutuallyexclusive in LUAD (left; n ¼ 493) and LUSC (right; n ¼ 490). Oncoprint (www.cbioportal.org): blue, high CN loss (GISTIC 2.0 threshold value of �2); red,high CN gain (GISTIC 2.0 threshold value of 2); green, mutations. E, SMARCA4 has the highest negative outlier sum statistics among SWI/SNF components (LUAD;n ¼ 598). F, histogram showing bimodal distribution of SMARCA4 gene expression (LUAD; n ¼ 548) highlighting the predicted patient "responder"population (red). G, protein expression and SMARCA4genomic annotation across lung cancer cell lines:mutation (�), copy-number loss (��), andgene silencing (���).
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and note that in addition to targeting SMARCA2/4, PFI-3 also hasactivity (�70% inhibition at 2 mmol/L) against the structurallyrelated fifth bromodomain from PBRM1, another SWI/SNFsubunit.
In cell-based chromatin-binding assays, using in situ cell extrac-tion techniques to remove non-chromatin bound proteins, weobserved dose-dependent displacement ofGFP-tagged SMARCA2bromodomain (i.e., 132 amino acid residues) by PFI-3 (Fig. 3Dand E). Notably, the inhibitor showed prolonged cell-targetengagement with similar potency (IC50 ¼ 5.78 mmol/L) fol-lowing 2 and 24 hours treatment (Supplementary Fig. S3). As anegative control, JQ1 did not inhibit the binding of ectopicallyexpressed SMARCA2 bromodomain, but selectively displacedGFP-tagged BRD4 (Fig. 3D, and data not shown). Takentogether, our cell-biochemical data cooperate the acceleratedfluorescence recovery after photobleaching (FRAP) reportedfor PFI-3 (35), and we conclude that PFI-3 is a selective,cell-permeable probe suitable to study the inhibition ofSMARCA2/4 bromodomains in cells.
PFI-3 does not phenocopy the growth-inhibitory effects ofSMARCA2 knockdown in lung cancer
Armed with PFI-3 and motivated by the context-specific phe-notype of SMARCA2 depletion (Fig. 2), we evaluated PFI-3 in theSMARCA4-deficient responder lines (A549, H1299, H157), butobserved no antiproliferative effects in either 3-day cell viability
(Fig. 4A) or long-term clonogenic assays (Fig. 4B and Supple-mentary Fig. S4). Because SWI/SNF is a multisubunit complexcontaining numerous chromatin-interacting domains, we specu-lated that selective SMARCA2 bromodomain inhibition by itselfis not sufficient to dislodge the endogenous SWI/SNF complexfrom chromatin. To elaborate on this, the binding of endogenous(full-length) SMARCA2 to chromatin was monitored by immu-nofluorescence in A549 cells using SMARCA2 knockdown as aspecificity control (Fig. 4C–E). Even high concentrations of PFI-3(30 mmol/L; 1 and 24 hours) were unable to displace theSMARCA2 protein from chromatin (Fig. 4C and D). Again, wecross-validated the in situ cell extraction assay using the referenceJQ1 inhibitor, which potently inhibited chromatin-binding ofendogenous BRD4 (Fig. 4C, bottom) but not SMARCA2 (data notshown). Taken together, these data suggest that the bromodo-main of SMARCA2 is dispensable for chromatin binding andSWI/SNF oncogenic activity in lung cancer.
PFI-3 treatment of synovial sarcoma cells and target genepromoter occupancy studies
As alterations in SWI/SNF have been implicated in diseaseprogression of synovial sarcomas (13), we also evaluated thepharmacologic activity of PFI-3 in Yamato and Aska cells. Thesecells harbor the hallmark recurrent chromosomal translocationt(X;18)(p11.2;q11.2), which fuses the SS18 gene, an integralsubunit of SWI/SNF complex, to one of the three SSX genes (SSX1,
Figure 2.SMARCA4-deficient lung cancer cellsselectively depend on SMARCA2. A,SMARCA2/4 protein levels in cell linesselected for RNAi studies. B and C,A549 (SMARCA4-deficient) and NCI-H460 (SMARCA4-proficient) cellstransduced with control (shLuc) orSMARCA2-targeting shRNAs (shS2)and assessed for knockdown (B) andcell viability (C) 1 week after puromycinselection (SD; n ¼ 6). D, clonogenicassay and crystal violet staining ofcolonies after 10 to 14 days. E, A549cells with inducible expression/reconstitution of full-length SMARCA4cDNA grown in the presence orabsence of doxycycline (�Dox) andanalyzed 4 and 7 days after doxycyclineinduction. F and G, followingdoxycycline treatment (5 days), A549cells were transfected with eithernonsilencing control (Ctrl) or SMARCA2siRNAs (S7 and S8) and evaluated forprotein knockdown (5 days aftertransfection; F) and clonogenicity (G).
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SSX2, and SSX4), observed in >95% of patients (36). Incorpo-ration of the SS18-SSX fusion protein into the SWI/SNF complexresults in eviction and degradation of the tumor-suppressorSMARCB1. The altered SWI/SNF complex binds to the SOX2locus, resulting in aberrant SOX2 expression, which is essentialfor proliferation of synovial sarcomas (13). Accordingly, YamatoandAska cells showhigh levels of Sox2 expression (37).Hence,wehypothesized that PFI-3may inhibit the altered SWI/SNF complexand impair cell growth, but we did not observe inhibition of cellproliferation in either 4-day viability (Fig. 5A) or long-termproliferation assays (Fig. 5B). We then assessed SOX2 expressionand found that PFI-3 treatment (day 3 and day 6) failed to reduceSOX2 transcript levels at pharmacologically relevant concentra-tions (Fig. 5C and Supplementary Fig. S5).
To elaborate on the lack of inhibitor-induced phenotype, weexamined SWI/SNF binding at the transcriptionally active SOX2promoter. SMARCA4 ChIP demonstrated high SWI/SNF complexoccupancy at the SOX2 promoter in Yamato cells, as previouslyreported (13), while no enrichment was observed at the MYOD1locus, a transcriptionally silent locus as confirmed by RNA Poly-merase II ChIP (Fig. 5D and Supplementary Fig. S5D). Impor-tantly, we also examined whether PFI-3 treatment could impair
binding of the SWI/SNF complex to the SOX2 locus, but observedonly a minor change in SOX2 promoter occupancy, suggestinginefficient inhibition of SWI/SNF binding (Fig. 5D). These dataare consistent with the in situ cell extraction results for PFI-3(Fig. 4C and D) showing that SMARCA2/4 bromodomain inhi-bition cannot displace the multisubunit SWI/SNF complex fromchromatin.
PFI-3 treatment of SMARCA4-dependent rhabdoid cancer orleukemia cells
Rhabdoid tumors are distinctly characterized by biallelic inac-tivation of SMARCB1, a core subunit of the SWI/SNF complex(10), and genetic studies have demonstrated that oncogenesismediated by SMARCB1 loss is dependent on the residual activityof SMARCA4-containing SWI/SNF complex (38). To establish abenchmark for PFI-3 treatment of A-204 and G-401 rhabdoidtumor cells, we identified two shRNAs that produced effective(>80%) SMARCA4 protein knockdown (Fig. 5E) and confirmedinhibition of cell viability in both short-term proliferation andlong-term clonogenic assays (Fig. 5F and G). In contrast with theRNAi phenotype, pharmacologic bromodomain inhibition didnot impact the growth of rhabdoid cancer cells (Fig. 5H).
Figure 3.PFI-3 is a potent, selective, and cellpermeable bromodomain inhibitor ofSMARCA2/4. A, chemical structure ofPFI-3 and biochemical potency(BROMOScan Kd's). B, BROMOScandose–response curves usingrecombinant purified bromodomains.C, PFI-3 selectivity (2 mmol/L) across 32bromodomains (DiscoverRx). D, in situcell extraction of HeLa cells expressingGFP-tagged SMARCA2 bromodomain(green) cotreated with SAHA (5 mmol/L)and PFI-3 (or DMSO control) for 2 hourswith Hoescht nuclear counterstain (red).HeLa control cells expressing GFP-tagged BRD4 treated (2 hours) with JQ1.E, displacement of the SMARCA2bromodomain from chromatin (IC50)quantified based on mean GFP signal pernucleus (SD; n ¼ 6).
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Finally, we extended our phenotypic evaluation of PFI-3 toleukemia as previous RNAi studies have shown that AML cellsdepend on SMARCA4 to support oncogenic transcriptional pro-grams (30, 31). Similar to our findings across lung, synovialsarcomas, and rhabdoid tumor cell lines, PFI-3 treatment did notafford an anticancer phenotype in THP-1 and MV4-11 leukemiccells (Supplementary Fig. S6) highlighting the critical importanceof pharmacologic drug target validation as a follow-up to RNAi-mediated knockdown studies.
Synthetic lethality of SMARCA2 knockdown is linked to thecatalytic ATPase activity
To genetically validate the PFI-3 results, we next used a 30UTRtargeting shRNA (shS2) to knockdown endogenous SMARCA2in H1299 cells engineered to express either wild-type (WT),ATP-binding pocket deficient (K755A; ref. 20) or bromodomainmutant (N1482W; ref. 39) forms of SMARCA2 (Fig. 6A). Ectopicexpression of either SMARCA2WT or the bromodomain binding-deficient mutant (BRD-Mut), but not the ATPase-dead form(ATP-Dead), completely rescued the RNAi-mediated LOF pheno-type (Fig. 6B and C). Likewise, A549 cells reconstituted withSMARCA4 WT or BRD-Mut (N1540W, Y1497F), but not ATP-Dead (K785A), were able to grow upon SMARCA2 knockdown(Fig. 6D–F).We also subjected the isogenicmatched-pair cell lines
to in situ cell extraction anddiscovered that the SMARCA2mutantsbound chromatin similarly to that of WT (Fig. 7A and B). Hence,failure of the ATP-Dead construct to rescue is due to lack ofcatalytic activity and not due to gross impairment in chromatinbinding. Altogether, our genetic assessment clearly demonstratesthat SMARCA4-deficient cancer cells do not require a functionalSMARCA2/4 bromodomain for growth. Instead, we unequivo-cally identify the catalytic activity of the ATPase domain as theappropriate, albeit more challenging, small-molecule drug target.
Genome-wide microarray analysis of SMARCA2/4 rescueexperiments
To examine the dependency on ATPase activity, we generatedmicroarray expression data (GSE69088) for the above cDNArescue experiments. Unsupervised clustering of the top variablegenes revealed three distinct expression profiles that were robustto the gene set size while clustering (Fig. 7C andD). In the absenceof SMARCA2 knockdown (shLuc), all H1299 derivative linesclustered together (Group 1) irrespective of the nature of theectopically expressed SMARCA2 constructs. On the other hand,the SMARCA2 knockdown cells (shSMARCA2) showed strongdifferential gene expression defining two distinct clusters: cellsrescued with either WT or BRD-Mut (Group 2) versus cellsexpressing either vector control (Ctrl) or ATP-Dead (Group 3).
Figure 4.Pharmacologic inhibition of SMARCA2/4 bromodomain in lung cancer. A,viability of SMARCA4-deficient (A549,H1299 and H157) or SMARCA4-proficient (H460) cells following PFI-3treatment (72 hours). Error bars, SD;n ¼ 3. B, A549 clonogenic assay (PFI-3and media replenished every three daysfor 1.5 weeks). C, in situ cell extraction(A549 cells) treated with PFI-3 or JQ1control for 2 hours followed byimmunofluorescence staining forendogenous, chromatin-boundbromodomain (green), and Hoeschtnuclear counterstain (red). D and E,immunofluorescence quantification (D)using SMARCA2 knockdown asspecificity control with correspondingimmunoblot confirmation (E). IF,immunofluorescence.
Targeting the SMARCA2/4 Bromodomain using RNAi and PFI-3
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Figure 5.Evaluation of PFI-3 in synovial sarcoma and rhabdoid tumor cells. A, viability of synovial sarcoma (Aska and Yamato) and HeLa cells treated with PFI-3(96 hours) relative to DMSO-treated controls (SEM; n ¼ 3). B, long-term (2-week) proliferation assay. Cells were split and replenished with freshmedia/PFI-3 every 3 or 4 days counting viable cells (SEM; n ¼ 3). C, PFI-3 treatment (3 days) does not repress SOX2 expression in Yamato cells.SOX2 transcript levels (RT-qPCR) normalized to GAPDH (SEM; n ¼ 12). D, control (DMSO) and PFI-3-treated Yamato cells (day 3) subjected to anti-SMARCA4 ChIP followed by qPCR for SOX2 promoter regions (target gene) or MYOD1 exon1 locus (negative control). The decrease in occupancy atthe SOX2 locus (10 mmol/L) is small but significant. � , P � 0.05 (SEM, n ¼ 9). E–G, A-204 and G-401 rhabdoid cells transduced with SMARCA4-targeting(shS4-4, shS4-5) or control (shLuc) shRNAs and analyzed for protein knockdown (1 week after puromycin selection; E), colony formation (2–3 weeksafter puromycin; F), and viability (CellTiter-Glo; 6 days after puromycin; G). Error bars, SD; n¼ 6. H, PFI-3 does not impair growth of G-401 cells (clonogenecity1.5 weeks; similar data for A-204 not shown). Media/PFI-3 was replenished every 3 days.
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Notably, expression/rescue using either SMARCA2 or SMARCA4showed identical clustering behavior, and differences in mRNAlevels within the three groups were nonsignificant. Hence, thetranscriptional profiles reinforce the view that BRD-Mut is able toperform similar functions to theWTgenewhileATP-Dead, despiteretaining its ability to bind chromatin (Fig. 7A and B), cannot.Consistent with the phenotypic responses (Fig. 6), gene setenrichment analysis (GSEA) revealed upregulation of apoptosisand death pathways in Group 3 versus the rescued cell lines(Group 2) highlighting the observed context-specific syntheticlethality (Supplementary Fig. S8).
The ATPase activity common between SMARCA2 and SMARC4shares a suppressive function on gene expression programs
Next, focusing on the requirement for ATPase activity, wecompared the SMARCA2 and SMARCA4 rescue profiles to see
whether similar gene expression programs may account for theobserved functional complementation. To establish a frame-work for this analysis, we first derived gene expression signa-tures for SMARCA4 expression in A549 cells (in the context ofSMARCA2 knockdown) comprising the top-100 upregulatedand downregulated genes, respectively (Supplementary TableS4). Using GSEA, we then looked for enrichment of thesesignatures with gene lists from SMARCA2 rescue experimentsas queries (Fig. 7E and F and Supplementary Table S5). Whencomparing ATP-Dead to WT, the enrichment profiles suggestthat the ATPase enzymatic activity preferentially reversesexpression of genes that are upregulated upon RNAi-mediated(synthetic lethal) knockdown of SMARCA2 (Fig. 7E). Genesdownregulated upon SMARCA2 knockdown were not fullyreversed by the rescue (Fig. 7F and Supplementary Table S5).Therefore, our microarray data clearly show that the ATPase
Figure 6.Rescue experiments highlight theimportance of ATPase activity forcancer-specific vulnerability. A,SMARCA2 cDNA rescue experiments inH1299 cells transduced with SMARCA2-targeting (50UTR) shRNA (shS2) orcontrol shRNA (shLuc) and analyzed byimmunoblotting 10 days afterpuromycin selection. B andC, in parallel,the isogenic cell lines were seeded in 6-well plates (24 hours after puromycinselection) and after 2 weeks stained bycrystal violet (B) and colony formingunits (CFU) were quantified (C). D,SMARCA4 cDNA rescue/reconstitutionexperiments in A549 cells transducedwith indicated shRNAs and analyzed byimmunoblotting 10 days afterpuromycin selection. E and F,clonogenic assay (crystal violetstaining; 1.5 weeks; E) andquantification of colony formingunits (SD; n ¼ 3; F).
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Targeting the SMARCA2/4 Bromodomain using RNAi and PFI-3
domain of SMARCA2/4 complements each other at the tran-scriptional level exerting similar suppressive function on spe-cific gene expression programs.
Next, using GSEA, we identified biologic pathways/statesshared by both rescue experiments leveraging annotated path-ways from the Molecular Signature Database (mSigDB; TheBroad Institute). GSEA identified enrichment of cell prolifera-tion (cell cycle, cyclin D1, G2–M checkpoint), chromatin remo-deling (mitotic spindle, DNA replication and synthesis), andtumorigeneis (EMT, integrin, KRAS and P53 pathway signa-tures). These gene expression programs, which are enrichedin rescued cells compared with cells lacking ATPase activitycommon to SMARCA2/4, have bonafide tumorigenic functions(Fig. 7G).
In conclusion, the SMARCA4 cDNA complementation (i.e., re-expression) and the SMARCA2 RNAi rescue experiments areconsistent with the observed lack of pharmacologic activity ofPFI-3 in SWI/SNF-mutant cancers, and we demonstrate for thefirst time that selective SMARCA2/4 bromodomain inhibition isnot a feasible therapeutic strategy for targeting aberrant SWI/SNFactivity in SWI/SNF-mutant cancers. Instead, drug discoveryefforts should be focused on inhibiting the ATPase catalyticactivity to deliver on the promise of robust, cancer-specific syn-thetic lethal therapy.
DiscussionA common theme has emerged from genetic studies where
imbalances between various paralogous subunits within SWI/SNF (e.g., SMARCA2/4 and ARID1A/B) can render cells moretumorigenic and simultaneously hypersensitive to targeting of theresidual complex (13, 20–23, 40, 41). However, despite the highprevalence of genomic lesions in SWI/SNF, studies have notaddressed how this observation can be translated into effective,drug discovery endpoints.
In this study, we first demonstrated context-specific antiproli-ferative phenotype of SMARCA2depletion in SMARCA4-deficientlung cancer using multiple, non-overlapping hairpins, as well asindependent siRNAs, validating the synthetic–lethal relationshipbetween SMARCA2 and SMARCA4 (20–22). We further showedthat expression of either SMARCA2 or SMARCA4 completelyrescued the effects of SMARCA2 knockdown in SMARCA4-defi-cient cells, indicating paralog dependence and reciprocal role ofthese two subunits in tumorigenesis. The functional complemen-tation of SMARCA2/4 was also evident at the transcriptional levelwhere the ATPase activity appears to control gene programsrelated to proliferation, cell cycle, and chromatin remodeling.Furthermore, genomic analysis revealed mutual exclusivity ofSMARCA2 and SMARCA4 mutations in LUSC and LUAD carci-noma and expression-based biomarker analysis outlined a
SMARCA4-deficient patient population predicted to dependexclusively on SMARCA2 activity (Fig. 1). Because SMARCA2-deficient mice are viable showing no overt phenotype (42), whileSMARCA4 inactivation is embryonic lethal (43), one mightanticipate a significant therapeutic window, if selective small-molecule SMARCA2 inhibitors can be developed that mimic theRNAi knockdown phenotype.
SMARCA2 contains an ATPase and bromodomain, suggestingat least two tractable avenues for inhibitor development. Target-ing the acetyl-lysine recognition function of SWI/SNF bromodo-mains (e.g., the SMARCA2/4, PBRM1, BRD7, and BRD9 subunits)represents an unexplored opportunity for perturbation of theSWI/SNF complex. Recently, the anticancer activity of BET bro-modomains inhibitors has fueled the development of novelchemical scaffolds that selectively target other bromodomains(6, 24, 25), and the PFI-3 inhibitor exemplifies one such novelchemical probe. However, despite being broadly available fromSGC, no phenotypic data have yet been reported. Hence, wesubjected PFI-3 to rigorous biochemical and cellular characteri-zation confirming its exquisite selectivity, potency and cell per-meability (Fig. 3). Such pharmacodynamics studies are a criticalcomponent of drug target validation studies as they providedconfidence that a compound-induced phenotype (or lack thereof)correlate with biochemical target engagement in cells.
Surprisingly, in contrast with SMARCA2 knockdown, PFI-3 didnot display any antiproliferative phenotype in SMARCA4-defi-cient lung cell lines across a variety of biologic assays. Likewise, inmodels harboring defined SWI/SNF alterations, including syno-vial sarcoma (SSX-fusion), rhabdoid tumors (SMARCB1-null),and leukemia (SMARCA4-dependent), PFI-3 did not mimic theanticancer phenotype observed upon RNAi-mediated knock-down of SMARCA2/4 (31, 38, 44). Mechanistically, and consis-tent with the lack of cellular phenotype, we discovered that PFI-3cannot displace endogenous SMARCA2 (i.e., lung) or SMARCA4(i.e., synovial sarcoma) from chromatin potentially due to theactivity of other chromatin-interacting SWI/SNF subunitshighlighting challenges in targeting large protein complexes. Thisresult is in sharp contrast with efficient chromatin displacement ofendogenous BRD4 by JQ1 (Fig. 4C), and we note the contrastingfeature of the BET family of tandem bromodomains, which arenot flanked by other known regulatory or conserved domains.
Recent studies have highlighted the role of residual SWI/SNFcomplex along with paralog dependence, indicating a potentialcombinatorial role of chromatin-interacting domains in SWI/SNFrecruitment (5). Our data further highlight the need to conductsimilar target identification/validation studies of other paralogsubunits like ARID1A and ARID1B that form mutually exclusiveSWI/SNF complexes and display a synthetic lethal relationship(40). Additional vulnerabilities like antagonism betweenSMARCB1 and EZH2, which renders rhabdoid tumors dependent
Figure 7.Chromatin binding and gene set enrichment highlights the importance of ATPase catalytic activity for cancer-specific vulnerability. A, immunofluorescentimages (H1299 cells) expressing either vector control or HA-tagged SMARCA2 wild-type, bromodomain-mutant or ATPase-dead constructs (red),and Hoechst counterstain (blue). B, quantification of chromatin binding (normalized to non-extracted immunofluorescent signal). C and D, clustering of1,000 most variable genes for SMARCA2 (C) and SMARCA4 rescue experiments (D). E and F, SMARCA2 knockdown signatures (derived from A549cells reconstituted with SMARCA4) comprising upregulated (up; E) and downregulated (down; F) genes. The ranked gene list (x-axis) was derivedfrom the SMARCA2 rescue experiments comparing ATP-Dead with WT as query. Genes responding differently (interaction contrast, Group 3 vs. Group 2)were ranked according to their P values with direction provided by the fold change. G, significantly enriched gene sets (mSigDB) shared betweenSMARCA2 and SMARCA4 focusing on their ATPase activity (i.e., WT vs. ATP-Dead cDNA expression).
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on EZH2 for disease maintenance (45), present another promis-ing approach to target SWI/SNF-mutant cancers. The antiproli-ferative response to SMARCA4 knockdown and EZH2 inhibitortreatment highlights clear dependencies on SWI/SNF activity (Fig.5E–Hand Supplementary Fig. S7A). However, it remains to beinvestigated whether other SWI/SNF-mutant cancers are sensitiveto EZH2 inhibition since we did not see activity of the EZH2inhibitor in SMARCA4-deficient lung cancer cells (SupplementaryFig. S7B).
For SWI/SNF-mutant cancers, our target validation approachhas focused on dissecting the functional contribution of animpaired SMARCA2/4 bromodomain or ATPase domain tocellular phenotype through parallel cDNA complementationand rescue experiments. The observation that expression ofBRD-Mut, but not ATP-Dead, can rescue the SMARCA2 knock-down phenotype is consistent with the pharmacologic PFI-3inhibitor data. Thus, our genetic and chemical findings con-verge and we unequivocally conclude that small-moleculeinhibition of the bromodomain is dispensable for the abilityof the SWI/SNF complex in controlling tumor growth. As such,the present study is the first to deprioritize SMARCA2/4 bro-modomain inhibition as a tractable target in genetically definedlung, synovial sarcoma, leukemia, and rhabdoid tumors. How-ever, we cannot exclude that compounds with a selectivityprofile that simultaneously inhibits additional bromodomainsin SWI/SNF (e.g., PBRM1, BRD7, and BRD9 subunits) could bean efficacious strategy (although pleiotropic bromodomaininhibition in normal cells could be a potential concern).Although the importance of ATPase activity has previouslybeen shown for chromatin remodeling (20), our studies pin-point the ATPase activity as the molecular synthetic-lethaltarget providing a genetically validated strategy for targetingSWI/SNF-mutant lung cancer.
ATPases represents a large and diverse family of proteins, manyof which perform chaperone-like functions assisting in the assem-bly, operation, and disassembly of protein complexes (46). Notsurprisingly, because numerous cellular processes are driven byenergy-dependent conformational changes in multisubunit com-plexes, ATPases have been implicated in various human diseaseswith several inhibitors in clinical use. However, most of these donot directly engage/bind the nucleotide-binding site (47). Devel-oping potent inhibitors that must compete with intracellularconcentrations of ATP (2–10 mmol/L) have been challenging.Phosphate groups contribute significant nucleotide-binding affin-ity, but to overcome poor cell-permeability of negatively-chargedphosphate groups, the majority of synthetic ATP analogues aredevoid of highly charged phospho-mimetic groups (47). More-over, the high sequence homology between ATP-binding sites(among ATPases and other ATP-binding proteins) represents aprofound selectivity challenge. Overall, there is a need for thedevelopment of novel, potent, and bioavailable ATPase inhibi-tors, and the successful design of ATP-competitive kinase inhibi-tors, yet another class of ATP-binding enzymes, supports at least inprincipal, the feasibility of targeting the ATP-binding site ofSMARCA2.
SMARCA2 belongs to the SNF2 family of chromatin remo-deling ATPases and contains most of the conserved motifsfound in SF2 helicases (48). However, SMARCA2/4 share littleoverall sequence homology with other helicases and even lesshomology with other ATPases (46, 48). Moreover, the sequencehomology of the SMARCA2 ATPase is limited to only two SNF2
clusters in the human genome— related SMARCA proteins anda class of DNA helicases, suggesting possibility for achievingexquisite selectivity over other ATPases. An obvious challengewould be to obtain selectivity over SMARCA4 as these enzymesare highly homogenous in their active site and dual inhibitionin normal cells could limit the therapeutic window (29).Structural insights often guide the design of selective inhibitors;however, very few X-ray crystal structures for ATPase domainsare currently available for SMARCA2/4-related proteins withmost being in open inactive conformation, like the yeast Chd1ATPase domain, highlighting the need for furthering structuralbiology. Another barrier for pharmaceutical development ofselective ATPase inhibitors is the current lack of commercialhigh-throughput screening assays and selectivity panels againstthe large family of ATP-binding proteins. Nevertheless, struc-tural diversity in the vicinity of the nucleotide-binding sites,including possible allosteric sites, should enable SMARCA2ATPase drug discovery supported by prior identification ofpotent and selective inhibitors of the ATPase activity of KIF11,Hsp90, and VCP (49, 50). The recent development of selectiveand cell-potent covalent inhibitors that block ATP binding, aswell as allosteric inhibitors that impair nucleotide turnover forthe VCP ATPase (50), is also an encouraging avenue for thedevelopment of inhibitors targeting the SMARCA2 ATPasecatalytic activity.
Taken together, our target validation studies identify theSMARCA2 ATPase domain, but not the bromodomain, as atractable, albeit more challenging therapeutic target for a well-defined SMARCA4-deficient patient population representingmore than 20,000 patients a year in the United States alone(i.e., 10%–20% of NSCLC cases). Moreover, the SMARCA4-deficient patient population generally lacks targetable oncogenes(such as mutant EGFR or ALK translocations; ref. 20), whichfurther emphasize the potential medical impact of developinginhibitors of the ATPase domain of SMARCA2/4.
Disclosure of Potential Conflicts of InterestT.A. Paul reports receiving commercial research grant from Pfizer, Inc. No
potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: B. Vangamudi, T.A. Paul, P.K. Shah, E. Leo,M. Peoples,T.P. Heffernan, D. Verhelle, P. Jones, S. Sharma, J.N. AndersenDevelopment ofmethodology:B.Vangamudi, T.A. Paul,M.Kost-Alimova,X. Shi,E. Leo, H.S. Mahadeshwar, T.N. Tieu, M. Peoples, S. Sharma, J.N. AndersenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Vangamudi, T.A. Paul, M. Kost-Alimova, L. Notte-baum, Y. Zhan, A. Protopopov, M. Peoples, A. Petrocchi, S. SharmaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):B. Vangamudi, T.A. Paul, P.K. Shah,M. Kost-Alimova,L. Nottebaum, E. Leo, H.S.Mahadeshwar, A. Protopopov, A. Futreal, C. Toniatti,D. Verhelle, P. Jones, J.N. AndersenWriting, review, and/or revision of the manuscript: B. Vangamudi, T.A. Paul,P.K. Shah, M. Kost-Alimova, L. Nottebaum, X. Shi, A. Futreal, T.P. Heffernan,J.R. Marszalek, C. Toniatti, A. Petrocchi, D. Verhelle, D.R. Owen, G.F. Draetta,P. Jones, W.S. Palmer, S. Sharma, J.N. AndersenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Vangamudi, T.A. Paul, Y. Zhan, G.F. DraettaStudy supervision: T.A. Paul, D. Verhelle, G.F. Draetta, S. Sharma, J.N. AndersenOther (synthesis of PFI-3 used for the studies aswell as obtained the profilingdata): W.S. PalmerOther (bioinformatics): P. Shah.
Cancer Res; 75(18) September 15, 2015 Cancer Research3876
AcknowledgmentsThe authors thank Dr. Daniel K. Treiber at DiscoveRx for custom assay
development, Drs. Norifumi Naka and Kazuyuki Itoh for the Aska and Yamatocells, and Dr. Chang-gong Liu for microarray services.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received January 5, 2015; revised May 31, 2015; accepted June 15, 2015;published OnlineFirst July 2, 2015.
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2015;75:3865-3878. Published OnlineFirst July 2, 2015.Cancer Res Bhavatarini Vangamudi, Thomas A. Paul, Parantu K. Shah, et al. Rescue and PFI-3 Inhibitor StudiesDrug Target in SWI/SNF-Mutant Cancers: Insights from cDNA The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a
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