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1Center for Research on Reproduction andWomen’s Health, University of Pennsylvania,Philadelphia, PA 19104, USA. 2Department of Obstetrics and Gynecology, West ChinaMedical School, Sichuan University, Chengdu 610041, China. 3Shanghai Key Laboratoryof Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital,Fudan University, Shanghai 200011, China. 4Department of Obstetrics and Gynecology,University of Pennsylvania, Philadelphia, PA 19104, USA. 5Department of RadiationOncology, University of Pennsylvania, Philadelphia, PA 19104, USA. 6Wistar Institute,Philadelphia, PA 19104, USA. 7Department of Pathology and LaboratoryMedicine, Uni-versity of Pennsylvania, Philadelphia, PA 19104, USA. 8Abramson Cancer Center, Uni-versity of Pennsylvania, Philadelphia, PA 19104, USA.*These authors contributed equally to this work.†Corresponding author. Email: linzhang@mail.med.upenn.edu

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Repression of BET activity sensitizes homologousrecombination–proficient cancers to PARP inhibitionLu Yang,1,2* Youyou Zhang,1* Weiwei Shan,1,3 Zhongyi Hu,1 Jiao Yuan,1 Jingjiang Pi,1

Yueying Wang,1 Lingling Fan,1,3 Zhaoqing Tang,1 Chunsheng Li,1,4 Xiaowen Hu,1,4 Janos L. Tanyi,4

Yi Fan,5 Qihong Huang,6 Kathleen Montone,7 Chi V. Dang,8 Lin Zhang1,4,8†

Strategies to enhance response to poly(adenosine diphosphate–ribose) polymerase inhibitor (PARPi) in primary andacquired homologous recombination (HR)–proficient tumors would be a major advance in cancer care. We used adrug synergy screen that combined a PARPi, olaparib, with 20 well-characterized epigenetic drugs and identifiedbromodomain and extraterminal domain inhibitors (BETis; JQ1, I-BET762, and OTX015) as drugs that acted syner-gistically with olaparib in HR-proficient cancer cells. Functional assays demonstrated that repressed BET activityreduces HR and thus enhances PARPi-induced DNA damage in cancer cells. We also found that inhibition or deple-tion of BET proteins impairs transcription of BRCA1 andRAD51, two genes essential for HR.Moreover, BETi treatmentsensitized tumors to PARP inhibition in preclinical animalmodels of HR-proficient breast and ovarian cancers. Finally,we showed that the BRD4 gene was focally amplified across 20 types of common cancers. Combination with BETicould greatly expand the utility of PARP inhibition to patients with HR-proficient cancer.

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INTRODUCTIONPoly[adenosine diphosphate (ADP)–ribose] polymerase inhibitors(PARPis) have been approved by the U.S. Food and Drug Adminis-tration (FDA) as monotherapy for women with germline BRCA1/2-mutated advanced ovarian cancer (1–10). They have also shownpromising clinical activity in breast (8, 10–12), prostate (8, 10, 13),and other solid cancers (8, 10), especially for tumors with defectiveDNA repair via homologous recombination (HR) (6, 7). PARP is anenzyme family that posttranslationally modifies its target proteins byconjugating polymeric chains ofADP-ribose [poly(ADP)-ribosylation(PARylation)] during a number of cellular processes including DNArepair, transcription, translation, cell signaling, and cell death (1–5, 14).Themolecular basis of PARPi effectiveness in cancer treatment appearsto mainly depend on the role of PARP, especially PARP1, in DNAdamage repair and transcriptional regulation (1–5).Multiplemechanismsof PARPi have been proposed, including suppression of base excisionrepair (1–5), trapping of PARP1 on damaged DNA (15), defectiveDNA repair protein recruitment (16), activation of nonhomologousend joining (NHEJ) (17) and alternative end joining (18, 19), andregulation of transcription (20, 21). HR-deficient cancer cells, suchas cells with BRCAmutations (6, 7) or PTEN loss (22), are extremelysensitive to PARPi. Despite promising clinical results (8–11), overcom-ing de novo and acquired HR proficiency is one of the major problemsfor PARPi (1–5). A large fraction of tumors are HR-proficient, suggest-ing that even in high-grade serous ovarian cancer, as many as 50% ofpatients may not benefit from this drug (1–5). Tumors that are initially

HR-deficient commonly acquire HR proficiency after PARPi treatmentby multiple mechanisms, including secondary mutations that restoreBRCA1/2 functions (23, 24), and loss of expression of PARP1 (25),53BP1 (26, 27), REV7 (28), or PTIP (29). Thus, the development ofstrategies to selectively impair HR in cancer cells and subsequentlysensitize HR-proficient cancers to PARP inhibition may providenew clinical applications (1–5). In this regard, drug combinationapproaches have been designed and evaluated in preclinical and earlyclinical trials (30–39).

Potent and selective small-molecule modulators influencingchromatin-modifying proteins have been developed as first-in-classtargeted therapies for patients with cancer (40, 41). For example,bromodomain (BRD) and extraterminal domain inhibitors (BETis)reversibly bind the BRDs of BET proteins and prevent their inter-action with acetylated histones and transcription factors (40, 41).Because well-ordered, deep, hydrophobic pockets in BRDs provide ahighly favorable locus for small-molecule compounds, a number ofBETis have been developed and evaluated in multiple preclinicalcancer models (42–54). BETis have advanced into clinical trials forcancer treatment, for example, in nuclear protein of the testis midlinecarcinoma (NMC) (55), acute leukemia (56), and multiple myeloma(57). BETis appear to preferentially affect the transcription of a rela-tively small subset of genes with super-enhancers (also called stretch-enhancers), such as MYC and BCL2 (42–49), suggesting that BETinhibition may specifically modulate the expression of a fractionof genes.

RESULTSDrug combination screen identifies BETi as actingsynergistically with PARPiTo explore whether modulation of epigenetic regulators can sensitizecancer cells to PARPi, we performed a drug combination screen in atriple-negative breast cancer cell line, MDA-MB-231, which expresseswild-type BRCA1/2 and modestly responds to PARPi. An FDA-approved PARPi (olaparib/AZD2281) and 20 well-characterized epige-netic drugs targeting seven classes of epigenetic regulators (>2 compoundsfor each class; table S1 and Fig. 1A) were chosen for the initial screen.

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Fig. 1. Drug combination screen identifies BETi as acting synergistically with PARPi. (A) The workflow of drug combination screen. IC50, median inhibitory con-centration. (B) Twenty compounds targeting seven classes of epigenetic modulators were examined in a combination screen with the PARPi, olaparib, in MDA-MB-231cells. The CI quantitatively depicts synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1). The color intensity shows the average of CI (red, synergism; green,antagonism). DZNep, 3-deazaneplanocin A. (C) Sensitivity of MDA-MB-231, OVCAR10, and VCaP to olaparib (Olap) alone, JQ1 alone, or olaparib combined with JQ1.Survival fraction (left) and the CI (right) are shown for each of these three cell lines. Fa, fraction affected. Error bars represent means ± SD. (D) Crystal violet staining ofanchorage-dependent colony formation assay indicates the sensitivity of cells to dimethyl sulfoxide (DMSO), JQ1, olaparib, or olaparib combined with JQ1. Effect oftreatments is shown for MDA-MB-231, OVCAR10, and VCaP cells. (E) Anchorage-independent soft agar assay results for MDA-MB-231, OVCAR10, and VCaP cells treatedwith DMSO, JQ1, olaparib, or olaparib combined with JQ1. Crystal violet staining of MDA-MB-231 and colony quantification results of all three cell lines are shown. Errorbars represent means ± SD. (F) Drug combination results (CI) for two PARPis (olaparib and veliparib) and three BETis (I-BET762, OTX015, and JQ1) examined in MDA-MB-231.

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The average combination index (CI) values of olaparib and each epi-genetic drug are summarized in Fig. 1B. Notably, all three BETis(JQ1, I-BET762, and OTX015) showed strong synergistic effects witholaparib in MDA-MB-231 cells. To confirm our observation, we vali-dated the above results in cell lines originating from three cancertypes, namely breast (MDA-MB-231), ovarian (OVCAR10), andprostate (VCaP) cancers, for which PARPi treatment has been pro-posed in clinical trials. Consistent with our initial screen, cell viabilityassays showed strong synergistic effects of JQ1 with olaparib in allthree cancer cell lines (Fig. 1C, all CI < 0.5). Then, we took a geneticapproach by simultaneously targeting BRD2, BRD3, and BRD4 usingpooled small interfering RNAs (siRNAs) and found that the pooledsiRNA treatment sensitized cells to PARP inhibition compared tothe control siRNA (fig. S1). Furthermore, the synergistic effect was alsoconfirmed by independent in vitro assays including anchorage-dependentcolony formation assay (Fig. 1D) and anchorage-independent soft agarassay (Fig. 1E). Furthermore, we expanded our drug combination toseveral well-characterized BETis (JQ1, I-BET762, and OTX015) andPARPis (olaparib and veliparib/ABT888) in MDA-MB-231 cells. Astrong synergistic effect was observed in all combinations tested inour study (Fig. 1F). Additionally, a synergistic effect of BETi and PARPicombination was observed in four HR-deficient cancer cell lines:UWB1.289, HCC1937, PEO1, and CAPAN-1 (fig. S2). We also foundthat JQ1 showed a synergistic effect in combination with the DNA-damaging drug cisplatin, but not with paclitaxel, which acts by stabiliz-ingmicrotubules inMDA-MB-231 cells (fig. S3). Finally, althoughBETirepresses the cell cycle (40, 41) and reduces the cancer stem cell popu-lation (58), the noncycling cancer cells and aldehyde dehydrogenase(ALDH)–positive cancer stem cells appeared insensitive to combinationtreatment with JQ1 and olaparib (figs. S4 and S5).

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

BET inhibition enhances PARPi-induced DNA damageTo test whether JQ1 enhances the DNA damage induced by PARPitreatment, we measured the amount of DNA damage induced byolaparib alone and in combination with JQ1 in HR-proficient cancercells using a comet assay. DNA damage was increased by olaparibalone in a dose-dependent manner, but the extent of DNA damagewas greatly increased when cells were treated with a combination ofJQ1 and olaparib (Fig. 2, A and B). Furthermore, we used immuno-fluorescence staining to measure the number of phosphorylated his-tone H2AX (gH2AX)–positive foci formed in the cells in response tovarious treatments. Consistent with previous results, the numbers ofgH2AX-positive foci were increased in cells subjected to combinationtreatment compared to cells treated with olaparib alone (Fig. 2, C andD). Finally, Western blot analysis also showed that the amounts ofgH2AX were increased in cells subjected to combination drug treat-ment compared to cells treated with olaparib alone (Fig. 2E).

BET inhibition reduces HRTo test whether BET inhibition reducesHR and subsequently enhancesPARPi response, we used an HR reporter assay (59) to monitor HRactivity in response to ionizing radiation (IR)–induced DNA damage(Fig. 3A) and found that JQ1 decreased HR in cancer cells (Fig. 3B).This result was further confirmed by the pooled siRNAs targetingBRD2, BRD3, and BRD4 (fig. S6). Then, we used a single-strandedDNA (ssDNA) staining assay (Fig. 3C) to detect the formation of IR-induced ssDNA, which is promoted by BRCA1 and acts as a substratefor HR repair (60). Consistently, ssDNA staining assays indicated thatJQ1 treatment reduces ssDNA formation in response to IR (Fig. 3D).Furthermore, immunofluorescence analyses indicated that JQ1 im-paired HR by decreasing the foci formation of BRCA1 and RAD51

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Fig. 2. BET inhibition enhances PARPi-induced DNA damage. (A) DNA damage in MDA-MB-231, OVCAR10, and VCaP cells treated with DMSO, JQ1, olaparib, or JQ1combined with olaparib, measured by the comet assay. Scale bars, 10 mm. (B) Extent of DNA damage, quantified by the tail moment in the comet assay. Statisticalanalysis by Student’s t test, *P< 0.05; n = 3. Bars representmean values of tail moment. (C) Representative images of gH2AX foci inMDA-MB-231, OVCAR10, and VCaP cells treatedwith DMSO, JQ1, olaparib, or JQ1 combined with olaparib. Scale bars, 10 mm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Quantification of the number of gH2AX-positive fociinMDA-MB-231, OVCAR10, and VCaP cells treatedwith DMSO, JQ1, olaparib, or JQ1 combinedwith olaparib. Statistical analysis by Student’s t test, *P < 0.05; n = 3. Error barsrepresent means ± SD. (E) Western blot analysis of gH2AX in MDA-MB-231, OVCAR10, and VCaP cells treated with DMSO, JQ1, olaparib, or JQ1 combined with olaparib.

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during IR-induced DNA damage (Fig. 3, E and F). Furthermore, weobserved that BET inhibition did not affect PARP1 trapping (fig. S7)and cellular PARylation (fig. S8). We also found that treatment withJQ1 increased NHEJ repair efficiency (fig. S9), suggesting that JQ1treatment may also increase the toxicity of PARPi by promoting error-prone NHEJ.

BET inhibition sensitizes HR-proficient tumors to PARPitreatment in vivoNext, we tested the effect of combined BETi and PARPi treatment inxenograft tumors. In an orthotopicMDA-MB-231 breast cancermodel,mice were randomly assigned to one of four groups to receive vehicle,JQ1, olaparib, or combination of olaparib and JQ1 (Fig. 4A). The com-bination of JQ1 and olaparib resulted in a significant inhibition oftumor growth (P < 0.0001; Fig. 4, B toD). Next, in anOVCAR10 ovar-ian cancer orthotopic model (Fig. 4E), as compared with the vehiclegroup, all treatment groups displayed slower growth of peritonealtumors and relief from the accumulation of ascites. Compared withsingle-agent treatment, combination of JQ1 and olaparib showed sig-nificantly decreased peritoneal ascites and tumor formation (P < 0.01;

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

Fig. 4, F to H). All treatment protocols were well tolerated, and meanbody weights were not significantly different among the four groups(fig. S10).

BET inhibition and depletion repress the expression ofBRCA1 and RAD51To investigate the molecular mechanism underlying the synergisticeffect between BETi and PARPi, we used microarray profiles gener-ated by Asangani et al. (50) to analyze transcriptome changes inducedby JQ1 treatment of VCaP cells. Gene ontology analysis indicated thatthese JQ1 response genes were significantly enriched in cancer-associatedpathways such as the DNA damage response (P = 0.00009; Fig. 5A).Notably, the expression of BRCA1 and RAD51, genes involved in theDNA damage repair via HR, was reduced about as much as that ofMYC, a well-demonstrated JQ1 target gene (Fig. 5B). We further vali-dated the above microarray results in two independent cell lines:MDA-MB-231 and OVCAR10. Real-time reverse transcription poly-merase chain reaction (RT-PCR) analysis showed that JQ1 treatmentreduced the mRNA expression of BRCA1 and RAD51 in a time- anddose-dependent manner (Fig. 5C). This result was confirmed at the

Fig. 3. BET inhibition reduces homologous recombination. (A) Schematic illustration of HR reporter assay. SD, splice donor; SA, splice acceptor; G, green; FP,fluorescent protein; ATG, the triplet code for the amino acid methionine. (B) HR-mediated DNA repair activity, measured by HR reporter assay, in MDA-MB-231 (left),OVCAR10 (middle), and VCaP (right). Statistical analysis by Student’s t test, *P < 0.05; n = 3. Error bars represent means ± SD. (C) Schematic illustration of ssDNA stainingassay. BrdU, 5-bromo-2′-deoxyuridine. (D) Representative images (left) and quantitative results (right) of IR-induced ssDNA foci formation in DMSO- or JQ1-treatedMDA-MB-231 cells. Cells without BrdU incorporation were used as negative control. Scale bars, 10 mm. (E and F) Representative images (left) and quantitative results(right) of IR-induced BRCA1 (E) and RAD51 (F) foci formation in DMSO- or JQ1-treated MDA-MB-231 cells. Scale bars, 10 mm. Ten gray (Gy) of IR was used for all three celllines. Statistical analysis by Student’s t test, *P < 0.05; n = 3 (D to F). Error bars represent means ± SD.

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protein level in MDA-MB-231 cells by Western blot (Fig. 5D). Thetumor samples collected from the in vivo treatment experimentshowed similar results (fig. S11). To test whether the repression ofBRCA1 and RAD51 by JQ1 was mediated by BET proteins, we tooka genetic approach by targeting each BET family member with twoindependent siRNAs. Knockdown specificity was confirmed by indi-vidual siRNA transfection (Fig. 5E). A reduction in both BRCA1 andRAD51 expression was observed when siRNAs targeting the threeBETs were pooled together (Fig. 5E). This result was further confirmedat the protein level in MDA-MB-231, OVCAR10, and VCaP cells byWestern blot (Fig. 5F). Notably, JQ1 treatment did not affect the ex-pression of other core proteins in the DNA damage repair pathways(fig. S12).

BETi directly represses transcription of BRCA1 and RAD51in cancer cellsTo explore the mechanism by which JQ1 represses the expression ofBRCA1 and RAD51, we analyzed chromatin immunoprecipitation(ChIP) with antibodies against BRD2/3/4 followed by ChIP sequencing(ChIP-seq) in VCaP cells treated with JQ1 (50). As expected, BRD2/3/4 immunoprecipitates shared overlapping enrichment for thepromoter regions of BRCA1 and RAD51, and JQ1 treatment reducedthe recruitment of all three proteins to these regions (Fig. 6, A andB). This observation was also validated by our ChIP–quantitativePCR (qPCR) analysis in MDA-MB-231 cells (Fig. 6, C and D). Next,we generated a luciferase reporter containing the DNA sequences ofthe BRCA1 or RAD51 promoter (Fig. 6, A and B). Consistent withChIP experiments, the luciferase activity of BRCA1 and RAD51 pro-moters was repressed by JQ1 treatment but was unchanged by olaparibtreatment. Compared to JQ1 treatment alone, there was no furtherreduction of the luciferase activity when the cells were treated witha combination of JQ1 and olaparib (Fig. 6, E and F). Furthermore,knocking down BRD2/3/4 by siRNAs genetically mimicked the effect

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of JQ1 in repressing the activity of BRCA1 and RAD51 promoters.Compared to the knockdown of individual BET family members,pooled siRNAs targeting all three BETs achieved greater repressionin the activity of BRCA1 and RAD51 promoters (Fig. 6, G and H).

We also explored potential super-enhancer regions in theBRCA1 and RAD51 loci using ChIP-seq analysis. ChIP-seq dataon H3K4me1, H3K4me3, and H3K27Ac indicated that a putativesuper-enhancer (a cluster of enhancers identified by high amountsof H3K27Ac/H3K4me1 and low amounts of H3K4me3) exists inthe downstream region of the BRCA1 gene in MDA-MB-231 cells(Fig. 7A). A consistent H3K27Ac binding pattern was also observedin VCaP cells, in which BRD2/3/4 were enriched in the same regions(Fig. 7B). JQ1 treatment markedly decreased the binding ability ofBRD2/3/4 in such enhancer regions in VCaP cells (Fig. 7B). Further-more, an identical H3K4me1, H3K4me3, and H3K27Ac binding pat-tern was also found in nonmalignant cells such as GM12878 cells(Fig. 7C), in which the BRCA1 gene and this putative enhancercluster are located in the same topologically associated domain, asdefined by high-resolution Hi-C analysis (Fig. 7D). To confirm thephysical interaction between the BRCA1 promoter and this potentialenhancer cluster, we performed quantitative analysis of chromosomeconformation capture assays (3C-qPCR) in three different cancer celllines. Among the seven pairs of primers designed to detect thepotential promoter-enhancer interaction (Fig. 7, A and E), the primersfor the promoter and the enhancer locus 2 showed the greatest inter-action strength and strongest response to JQ1 treatment in all threelines (Fig. 7E). The size of the corresponding PCR product was con-firmed by electrophoresis (Fig. 7F). The above observations were alsovalidated by ChIP-qPCR analysis in MDA-MB-231 cells (Fig. 7G). Astrong transcriptional enhancing activity was observed in the cellstransfected with the BRCA1 promoter-enhancer vector compared tothe BRCA1 promoter vector, and the enhancer activity was inhibitedby JQ1 alone or the combination of JQ1 and olaparib but not by

Fig. 4. BET inhibition sensitizes HR-proficient tumors to PARPi treatment in vivo. (A) Schematic illustrating the MDA-MB-231 mouse xenograft experimentaldesign. MDA-MB-231 cells were implanted in the mammary fat pad and grown until tumors reached a size of about 30 mm3. Xenografted mice were randomized and thenreceived vehicle, JQ1 (50mg/kg), olaparib (50mg/kg), or the combination of both agents as indicated (5 days a week for 3 weeks). Calipermeasurements were taken every 3 daysafter the initiation of drug treatment. (B) Mean tumor volume is shown. Statistical analysis by Student’s t test. Error bars representmeans ± SD. (C) Images of tumors collected fromanimals receiving vehicle, JQ1, olaparib, or the combination of both agents. (D) Individual tumor weights from different treatment groups are shown. Statistical analysis byStudent’s t test. Error bars represent means ± SD. (E) Schematic illustrating the OVCAR10 mouse xenograft experimental design. OVCAR10 cells were implanted intraperitoneallyin mice and grown for 2 weeks. Xenografted mice were randomized and then received vehicle, JQ1 (50 mg/kg), olaparib (50 mg/kg), or the combination of both agents asindicated (5 days a week for 4 weeks). (F) Images of the tumor nodes (diameter, >0.5 mm) collected from mice receiving different treatments. (G and H) Quantification of theweight of tumor nodules (G) and the volume of ascites (H) in mice receiving different treatments. Statistical analysis by Student’s t test. Error bars represent means ± SD.

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olaparib alone (Fig. 7H). Finally, we confirmed the above result bysiRNAs specifically targeting each BET family member (Fig. 7I).

Recurrent copy number amplification of the BRD4 gene isobserved across common cancersTo characterize the genomic alterations of BET genes in cancer, weretrieved RNA sequencing (RNA-seq; n = 8298), single-nucleotide

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

polymorphism (SNP) microarray (n = 9445), and exon sequencing(exon-seq; n = 8899) profiles across 20 common cancer types (table S2)from The Cancer Genome Atlas (TCGA). First, we found that genefusion is a rare genomic alteration for BET genes across all cancertypes. Only 10 fusion events (8 from BRD4 and 2 from BRD3) wereidentified across 8298 RNA-seq profiles of TCGA tumor specimens(Fig. 8A and table S3). No recurrent fusion events were identified.

Fig. 5. BET inhibition and depletion repress the expression of BRCA1 and RAD51. (A) Pathways overrepresented by JQ1 response genes (fold change, >2) in VCaPcells according to ConsensusPathDB analysis on the basis of gene ontology terms (left). Diagram depicts pathway interactions, grouped by pathways (right). Line thicknesscorresponds to the number of shared proteins between two pathways. Circle size corresponds to the number of JQ1 response genes in each pathway. The color intensityof circle indicates the P value for each pathway. miRNA, microRNA. (B) RNA expression of BRCA1, RAD51, and MYC in two independent microarray experiments in VCaP cellstreated with DMSO or JQ1 (500 nM for 24 hours). (C) BRCA1 and RAD51 mRNA expression measured by real-time RT-PCR in MDA-MB-231 cells (left) and OVCAR10 cells(right) treated with 500 nM JQ1 for different time periods or with different concentrations of JQ1 for 72 hours. Error bars represent means ± SD. (D) BRCA1 and RAD51expression assessed by Western blot in MDA-MB-231 cells treated with 500 nM JQ1 for different time periods (top) or with different concentrations of JQ1 for 72 hours(bottom). Tubulin and actin served as loading controls. (E) BRD2/3/4, BRCA1, and RAD51 mRNA expression in MDA-MB-231 cells treated with individual BRD2/3/4 siRNAs orwith BRD2/3/4 siRNA pools. Error bars represent means ± SD. (F) BRD2/3/4, BRCA1, RAD51, and MYC expression measured by Western blot in cells treated with individualBRD2/3/4 siRNAs or with a BRD2/3/4 siRNA pool. Left: MDA-MB-231. Middle: OVCAR10. Right: VCaP. Actin served as a loading control.

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Second, we found that there was no recurrent mutation observed inBET genes, except for a low-frequency recurrent BRD2mutation in co-lon cancer (3.01%) and BRDT in pancreatic (0.55%) and endometrial(8.12%) cancers, across 20 cancer types (n = 8899) based on exon-seqanalysis (table S4). Last, we analyzed the somatic copy number altera-tions (SCNAs) of BET genes in cancer via SNP microarray analysis of9445 tumors in 20 cancer types from TCGA. In a pan-cancer analysis,recurrent amplification of BRD4 was observed (Fig. 8, B and C).Analyses for each individual cancer type showed that BRD4 was re-currently amplified in breast, liver, ovarian, and endometrial cancers(Fig. 8, B and D). By contrast, BRD3 was recurrently deleted in breastand ovarian cancers at a low frequency (Fig. 8B). Although the BRDTgene (coding a testis-specific BET protein) was recurrently deleted andmutated in six and two cancer types, respectively, the RNA-seq analysisindicated that BRDTmRNAwas not detectable in any cancer type (Fig.8D). This suggests that deletion and mutation of BRDT may be a pas-senger event. In contrast, BRD2, BRD3, and BRD4 were widelyexpressed in all cancer types examined in our study (Fig. 8E). We founda positive correlation between BRD4 copy number and gene expressionacross all cancer types and in individual cancers (Fig. 8F). Cancer typesthat had higher expression of BRD4 SCNAs (such as ovarian and breastcancers) demonstrated strongerRNA-SCNAcorrelations than the cancertypes with fewer SCNAs (such as prostate cancer) (Fig. 8F). We alsofound that theBRD4-amplified cancer cell lines have decreased sensitivityto PARPi (fig. S13). The recurrent focal amplification of the BRD4 geneprovides a strong rationale for BRD4-targeted therapy for cancer patients.

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DISCUSSIONStrategies that selectively disrupt HR in cancer cells may sensitizeHR-proficient tumors to PARP inhibition. Furthermore, given thatacquiredHRproficiency isoneof themost commonresistancemechanismsto PARP inhibition, such approaches may also overcome resistance toPARPi treatment, which can develop in tumors initially sensitive toPARPi. In this regard, several approaches have been evaluated inpreclinical and early clinical trials (30–39), such as combinations ofPARPi with a CDK1 inhibitor that represses phosphorylation ofBRCA1 (30) and combination of PARPiwith PI3K (phosphatidylinositol3-kinase)/AKT inhibitors that down-regulates BRCA1/2 expression (31)and suppresses RAD51 foci formation (32). Using a drug synergy screen,we identified BETi as acting synergistically with PARPi in HR-proficientcells. Our results indicate that BETimay synergize with PARPi to treatpatients with de novo HR-proficient cancer that is primarily resistantto PARPi because repressed BET activity impairs BRCA1 and RAD51expression, subsequently converting HR-proficient tumors to HR-deficient tumors (fig. S14A). Furthermore, our results suggest thatBETi may resensitize tumors with acquired HR proficiency to PARPitreatment, thus overcoming PARPi resistance. BETi may act byrepressing the expression of secondaryBRCA1mutations that restoresBRCA1 function (fig. S14B) or blocking the expression of BRCA1 andRAD51 in the context of other resistance mechanisms (such as 53BP/REV7 loss and BRCA2 reversion mutation) to overcome PARPiresistance. Because BET inhibition simultaneously represses the ex-pression of BRCA1 and RAD51, BETi may also enhance the response

Fig. 6. JQ1 directly represses the promoter activities of BRCA1 and RAD51. (A and B) BRD2/3/4 binding pattern in the BRCA1 (A) or RAD51 (B) promoter regions inVCaP cells treated with DMSO or JQ1. Bottom: Illustrations of BRCA1 promoter–luciferase (Luc) (A) and RAD51 promoter–Luc (B) reporters, in which BRCA1 or RAD51promoter containing the BRD2/3/4 binding region was cloned into pGL3. (C and D) Quantification of the amount of BRCA1 (C) or RAD51 (D) promoter bound toBRD2/3/4 in MDA-MB-231 cells treated with DMSO or JQ1. Statistical analysis by Student’s t test, *P < 0.05; n = 3. Error bars represent means ± SD. IgG, immunoglobulinG. (E and F) Luciferase reporter assay of the activities of the BRCA1 (E) and RAD51 (F) core promoter reporters in MDA-MB-231, OVCAR10, and VCaP cells treated with DMSO,JQ1, olaparib, or JQ1 combined with olaparib. Statistical analysis by Student’s t test, *P < 0.05; n = 3. Error bars represent means ± SD. (G and H) Luciferase reporter assay ofthe activities of the BRCA1 (G) and RAD51 (H) core promoter reporters in MDA-MB-231, OVCAR10, and VCaP cells treated with individual siRNAs targeting BRD2/3/4 orpooled siRNAs. Statistical analysis by Student’s t test, *P < 0.05; n = 3. Error bars represent means ± SD.

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to PARP inhibition inHR-deficient tumors such asBRCA1- or BRCA2-mutated cancers by further blocking RAD51 expression (fig. S14C). Fi-nally, given that deficiency of HR increases the sensitivity of cancer cellsto treatment with DNA damage agents, BETi may also act synergisti-cally with classical chemotherapy drugs. Our study provides a strongrationale for clinical application of BETi in combination with PARPior other DNA-damaging anticancer agents.

Themolecularmechanismof BET inhibition in cancer treatment isa fundamental question. Considering the critical role of BET proteinsin gene transcriptional elongation, the mechanism underlying theantitumor activity of BETimay bemainly mediated by transcriptional

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

repression (40, 41). The observation that BET inhibition only repressesthe transcription of a selected subset of genes was unexpected, indicat-ing that BETi may specifically impair the expression of a fraction ofcancer-associated genes such as MYC (42–49). We showed that expres-sion ofBRCA1 andRAD51was repressed byBETi to a comparable extentas MYC repression in multiple cancer models. A potential super-enhancer of BRCA1was identified in epithelial cancer cells, and it is con-served in nontransformednormal cells.Wedemonstrated that repressionof BETproteins impairs expression ofBRCA1 andRAD51, subsequentlydisrupting HR in tumor cells. Our study thus provides a mechanisticinsight into the development of BET-targeted therapy in cancer.

Fig. 7. JQ1 represses the enhancer-promoter interaction of BRCA1. (A) H3K27Ac, H3K4me1, H3K4me3 profiles in MDA-MB-231. Right and left rectangle frame areasindicate the BRCA1 promoter and enhancer locus 2, respectively, which were used for the BRCA1 promoter-enhancer reporter construct. Upper schematic illustrationindicates the construction of the BRCA1 promoter-enhancer reporter. Lower schematic illustrates the Xba I restriction sites in BRCA1 promoter and enhancer loci. P inred indicates promoter, and E in black indicates enhancer. Numbers 1 to 7 indicate seven Xba I restriction sites in the enhancer locus. The position of the BRCA1 geneis indicated below. (B) H3K27Ac in VCaP cells (top) and BRD2/3/4 profiles in VCaP treated with DMSO or JQ1 (bottom). (C) H3K27Ac, H3K4me1, H3K4me3, andCCCTC-binding factor (CTCF) profiles in GM12878. (D) A BRCA1 topologically associated domain (TAD) region (indicated by a thick black bar) was predicted on thebasis of the Hi-C data. Normalized Hi-C interacting frequencies were shown as a triangle-like two-dimensional heat map, which was overlaid on ChIP-seq data (C) inGM12878 cells. Red indicates the intensity of the interacting frequencies of the Hi-C data. Chr 17, chromosome 17. (E) 3C-qPCR analysis of long-distance interactionsbetween the BRCA1 promoter and seven enhancer loci [locus 1 to 7 indicated in (A)]. The relative amount of ligation product between the BRCA1 promoter and eachof the seven different enhancer loci was plotted. Results from MDA-MB-231, OVCAR10, and VCaP cells treated with DMSO or JQ1 are shown. The data were normal-ized to a GAPDH loading control. (F) PCR products from the BRCA1 promoter and enhancer locus 2 (E2, top) and from the BRCA1 promoter and enhancer locus 6 (E6,bottom). Ligation sample from bacterial artificial chromosome (BAC) was used as a positive control. No specific PCR product was found in the ligation sample fromthe BRCA1 promoter and enhancer locus 6, even when the BAC control band was overexposed (shown in red). (G) Quantification of the amount of BRCA1 enhancerlocus 2 bound to BRD2/3/4 in MDA-MB-231 cells treated with DMSO or JQ1. (H) Luciferase reporter assay of BRCA1 enhancer activity in MDA-MB-231, OVCAR10, andVCaP cells treated with DMSO, JQ1, olaparib, or JQ1 combined with olaparib. (I) Luciferase reporter assay of BRCA1 enhancer activity in MDA-MB-231, OVCAR10, andVCaP cells treated with BRD2/3/4 siRNA.

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Our study has been limited to focusing on the BETi targeting genes,which are directly involved inDNAdamage repair. Althoughwe believethat direct transcriptional repression of HR genes is the dominantmechanism, we cannot exclude other indirect mechanisms that may alsocooperate or contribute to this synergistic effect. For example, BETi re-presses expression ofMYC (42–49), a transcriptional amplifier, and thus,MYC reduced by BETi might also indirectly contribute to reduction ofHR gene expression and enhancement of NHEJ activity, especially inthe MYC-hyperactivated tumors. Additionally, BETi may also directlyinfluence the DNA damage response by interacting with chromatinremodeling complexes. A recent study identified a short isoform ofBRD4, which serves as an insulator of chromatin, modulating thesignaling response to DNA damage (61). Therefore, it is still necessaryto further characterize how othermechanisms cooperate or contribute tothe BETi/PARPi synergistic effect in some cellular contexts. Despitethese limitations, our findings provide a strong rationale for clinicalapplication of PARPi in the setting of combination with BETi to treatcancers with de novo or acquired resistance to PARP inhibition.

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

MATERIALS AND METHODSStudy designThe overall goal of this study was to identify epigenetic modulatorsthat can act synergistically with PARPis in preclinical models of humancancer. Further experiments were designed to characterize the molecu-lar and cellularmechanisms of the combination. The selection of cancercell lines was based on the clinical application of PARPi: We chose celllines originating from ovarian (OVCAR10), breast (MDA-MB-231),and prostate (VCaP) cancers, for which PARPi treatment has beenapplied or proposed in clinic. Given that olaparib (AZD2281) was thefirst PARPi approved by FDA, we chose it in our initial screen. For allxenograft studies, sample sizes were decided using data from prelimi-nary caliper measurements of tumor growth. We performed prelimi-nary experiments using a few mice per group to decide the samplesize, followed by larger studies to perform the in vivo drug combinationexperiments with statistical analysis. We also repeated experimentsto ensure reproducibility. Xenografted mice were randomized beforereceiving different treatments. The resulting tumors were analyzed in

Fig. 8. Recurrent copy number amplification of BRD4 gene was observed across common cancers. (A) Fusions of BET genes in TCGA samples are shown by a Circosplot. The fused genes are illustrated as a line that connects two parental genes. Line thickness corresponds to FFPM (fusion fragments per million total RNA-seq reads)value. (B) Plot of the recurrent copy number alterations in BET genes across 20 cancer types. Red and blue indicate amplification and deletion, respectively. Dot sizeindicates the G score, which represents frequency and amplitude of copy number alteration. (C) Significance of recurrent BRD4 amplification across 20 types of cancers.GISTIC q values (x axis) for amplifications are plotted across the genomic locus harboring the BRD4 gene (y axis). (D) Copy number profiles of the BRD4 locus from breast,ovarian, and prostate tumor specimens. Each sample is represented with a vertical line, and the positions of BRD4 are noted with black horizontal lines. Red, gain; blue, loss.(E) Heat map of the 50th percentile of BET gene mRNA expression across cancers. Each cancer type is represented by a column, and each BET gene is represented by a row.The intensity of blue indicates mRNA expression. FPKM, fragments per kilobase of exon per million. (F) A positive correlation between BRD4 gene copy number(CN) and RNA expression was observed in cancer specimens.

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a blinded manner. For all in vitro experiments, no randomization orblinding was used because they were deemed unfeasible because of thenature of the performed experiments.

Statistical analysisStatistical analysis was performed using SPSS andR software. All resultsare expressed as means ± SD, and P < 0.05 indicates significance.

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/400/eaal1645/DC1Materials and MethodsFig. S1. BET depletion increases PARPi sensitivity.Fig. S2. BETi synergizes with PARPi in HR-deficient cancer cell lines.Fig. S3. BETi synergizes with cisplatin but not paclitaxel in MDA-MB-231 cells.Fig. S4. Combination treatment with BETi and PARPi does not act synergistically in noncyclingcancer cells.Fig. S5. BET inhibition decreases the ALDH+ tumor-initiating cell population.Fig. S6. BET depletion decreases HR.Fig. S7. BET inhibition does not affect PARP trapping.Fig. S8. BET inhibition does not affect total cellular PARylation and Ku80 PARylation.Fig. S9. BET inhibition increases NHEJ repair efficiency.Fig. S10. Combining JQ1 and olaparib does not increase toxicity to mice.Fig. S11. BET inhibition represses the expression of BRCA1 and RAD51 in vivo.Fig. S12. The effect of BET inhibition on the expression of other proteins was analyzed byWestern blot.Fig. S13. BRD4 amplification results in PARPi resistance.Fig. S14. The study provides a strong rationale for clinical application of PARPi in combinationwith BETi.Table S1. Epigenetic compounds used in drug combination screen (provided as an Excel file).Table S2. TCGA specimens used in this study (provided as an Excel file).Table S3. Fusion events of BET genes in TCGA (provided as an Excel file).Table S4. Mutation frequency of BET genes in TCGA (provided as an Excel file).

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REFERENCES AND NOTES1. C. J. Lord, A. N. J. Tutt, A. Ashworth, Synthetic lethality and cancer therapy: Lessons

learned from the development of PARP inhibitors. Annu. Rev. Med. 66, 455–470 (2015).2. C. L. Scott, E. M. Swisher, S. H. Kaufmann, Poly (ADP-ribose) polymerase inhibitors: Recent

advances and future development. J. Clin. Oncol. 33, 1397–1406 (2015).3. P. A. Konstantinopoulos, R. Ceccaldi, G. I. Shapiro, A. D. D’Andrea, Homologous

recombination deficiency: Exploiting the fundamental vulnerability of ovarian cancer.Cancer Discov. 5, 1137–1154 (2015).

4. F. Y. Feng, J. S. de Bono, M. A. Rubin, K. E. Knudsen, Chromatin to clinic: The molecularrationale for PARP1 inhibitor function. Mol. Cell 58, 925–934 (2015).

5. Y. Pommier, M. J. O’Connor, J. de Bono, Laying a trap to kill cancer cells: PARP inhibitorsand their mechanisms of action. Sci. Transl. Med. 8, 362ps17 (2016).

6. H. E. Bryant, N. Schultz, H. D. Thomas, K. M. Parker, D. Flower, E. Lopez, S. Kyle, M. Meuth,N. J. Curtin, T. Helleday, Specific killing of BRCA2-deficient tumours with inhibitors ofpoly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).

7. H. Farmer, N. McCabe, C. J. Lord, A. N. J. Tutt, D. A. Johnson, T. B. Richardson,M. Santarosa, K. J. Dillon, I. Hickson, C. Knights, N. M. B. Martin, S. P. Jackson, G. C. M. Smith,A. Ashworth, Targeting the DNA repair defect in BRCA mutant cells as a therapeuticstrategy. Nature 434, 917–921 (2005).

8. P. C. Fong, D. S. Boss, T. A. Yap, A. Tutt, P. Wu, M. Mergui-Roelvink, P. Mortimer,H. Swaisland, A. Lau, M. J. O’Connor, A. Ashworth, J. Carmichael, S. B. Kaye, J. H. M. Schellens,J. S. de Bono, Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutationcarriers. N. Engl. J. Med. 361, 123–134 (2009).

9. M. W. Audeh, J. Carmichael, R. T. Penson, M. Friedlander, B. Powell, K. M. Bell-McGuinn,C. Scott, J. N. Weitzel, A. Oaknin, N. Loman, K. Lu, R. K. Schmutzler, U. Matulonis,M. Wickens, A. Tutt, Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients withBRCA1 or BRCA2 mutations and recurrent ovarian cancer: A proof-of-concept trial.Lancet 376, 245–251 (2010).

10. B. Kaufman, R. Shapira-Frommer, R. K. Schmutzler, M. W. Audeh, M. Friedlander,J. Balmaña, G. Mitchell, G. Fried, S. M. Stemmer, A. Hubert, O. Rosengarten, M. Steiner,N. Loman, K. Bowen, A. Fielding, S. M. Domchek, Olaparib monotherapy in patients withadvanced cancer and a germline BRCA1/2 mutation. J. Clin. Oncol. 33, 244–250 (2015).

11. A. Tutt, M. Robson, J. E. Garber, S. M. Domchek, M. W. Audeh, J. N. Weitzel, M. Friedlander,B. Arun, N. Loman, R. K. Schmutzler, A. Wardley, G. Mitchell, H. Earl, M. Wickens,

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

J. Carmichael, Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1or BRCA2 mutations and advanced breast cancer: A proof-of-concept trial. Lancet376, 235–244 (2010).

12. H. S. Rugo, O. I. Olopade, A. DeMichele, C. Yau, L. J. van’t Veer, M. B. Buxton, M. Hogarth,N. M. Hylton, M. Paoloni, J. Perlmutter, W. F. Symmans, D. Yee, A. J. Chien, A. M. Wallace,H. G. Kaplan, J. C. Boughey, T. C. Haddad, K. S. Albain, M. C. Liu, C. Isaacs, Q. J. Khan, J. E. Lang,R. K. Viscusi, L. Pusztai, S. L. Moulder, S. Y. Chui, K. A. Kemmer, A. D. Elias, K. K. Edmiston,D. M. Euhus, B. B. Haley, R. Nanda, D. W. Northfelt, D. Tripathy, W. C. Wood, C. Ewing, R. Schwab,J. Lyandres, S. E. Davis, G. L. Hirst, A. Sanil, D. A. Berry, L. J. Esserman; I-SPY 2 Investigators,Adaptive randomization of veliparib–carboplatin treatment in breast cancer. N. Engl. J. Med.375, 23–34 (2016).

13. J. Mateo, S. Carreira, S. Sandhu, S. Miranda, H. Mossop, R. Perez-Lopez, D. Nava Rodrigues,D. Robinson, A. Omlin, N. Tunariu, G. Boysen, N. Porta, P. Flohr, A. Gillman, I. Figueiredo,C. Paulding, G. Seed, S. Jain, C. Ralph, A. Protheroe, S. Hussain, R. Jones, T. Elliott,U. McGovern, D. Bianchini, J. Goodall, Z. Zafeiriou, C. T. Williamson, R. Ferraldeschi,R. Riisnaes, B. Ebbs, G. Fowler, D. Roda, W. Yuan, Y.-M. Wu, X. Cao, R. Brough,H. Pemberton, R. A’Hern, A. Swain, L. P. Kunju, R. Eeles, G. Attard, C. J. Lord, A. Ashworth,M. A. Rubin, K. E. Knudsen, F. Y. Feng, A. M. Chinnaiyan, E. Hall, J. S. de Bono,DNA-repair defects and olaparib in metastatic prostate cancer. N. Engl. J. Med. 373,1697–1708 (2015).

14. Y. Hu, S. A. Petit, S. B. Ficarro, K. J. Toomire, A. Xie, E. Lim, S. A. Cao, E. Park, M. J. Eck,R. Scully, M. Brown, J. A. Marto, D. M. Livingston, PARP1-driven poly-ADP-ribosylationregulates BRCA1 function in homologous recombination–mediated DNA repair.Cancer Discov. 4, 1430–1447 (2014).

15. J. Murai, S.-y. N. Huang, B. B. Das, A. Renaud, Y. Zhang, J. H. Doroshow, J. Ji, S. Takeda,Y. Pommier, Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res.72, 5588–5599 (2012).

16. M. Li, X. Yu, Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation.Cancer Cell 23, 693–704 (2013).

17. A. G. Patel, J. N. Sarkaria, S. H. Kaufmann, Nonhomologous end joining drivespoly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proc. Natl. Acad. Sci. U.S.A. 108, 3406–3411 (2011).

18. P. A. Mateos-Gomez, F. Gong, N. Nair, K. M. Miller, E. Lazzerini-Denchi, A. Sfeir,Mammalian polymerase q promotes alternative NHEJ and suppresses recombination.Nature 518, 254–257 (2015).

19. R. Ceccaldi, J. C. Liu, R. Amunugama, I. Hajdu, B. Primack, M. I. R. Petalcorin, K. W. O’Connor,P. A. Konstantinopoulos, S. J. Elledge, S. J. Boulton, T. Yusufzai, A. D. D’Andrea,Homologous-recombination-deficient tumours are dependent on Polq-mediated repair.Nature 518, 258–262 (2015).

20. J. C. Brenner, B. Ateeq, Y. Li, A. K. Yocum, Q. Cao, I. A. Asangani, S. Patel, X. Wang, H. Liang,J. Yu, N. Palanisamy, J. Siddiqui, W. Yan, X. Cao, R. Mehra, A. Sabolch, V. Basrur,R. J. Lonigro, J. Yang, S. A. Tomlins, C. A. Maher, K. S. J. Elenitoba-Johnson, M. Hussain,N. M. Navone, K. J. Pienta, S. Varambally, F. Y. Feng, A. M. Chinnaiyan, Mechanisticrationale for inhibition of poly(ADP-ribose) polymerase in ETS gene fusion-positive prostatecancer. Cancer Cell 19, 664–678 (2011).

21. M. J. Schiewer, J. F. Goodwin, S. Han, J. C. Brenner, M. A. Augello, J. L. Dean, F. Liu,J. L. Planck, P. Ravindranathan, A. M. Chinnaiyan, P. McCue, L. G. Gomella, G. V. Raj,A. P. Dicker, J. R. Brody, J. M. Pascal, M. M. Centenera, L. M. Butler, W. D. Tilley,F. Y. Feng, K. E. Knudsen, Dual roles of PARP-1 promote cancer growth andprogression. Cancer Discov. 2, 1134–1149 (2012).

22. K. J. Dedes, D. Wetterskog, A. M. Mendes-Pereira, R. Natrajan, M. B. Lambros,F. C. Geyer, R. Vatcheva, K. Savage, A. Mackay, C. J. Lord, A. Ashworth, J. S. Reis-Filho, PTENdeficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARPinhibitors. Sci. Transl. Med. 2, 53ra75 (2010).

23. S. L. Edwards, R. Brough, C. J. Lord, R. Natrajan, R. Vatcheva, D. A. Levine, J. Boyd,J. S. Reis-Filho, A. Ashworth, Resistance to therapy caused by intragenic deletion inBRCA2. Nature 451, 1111–1118 (2008).

24. W. Sakai, E. M. Swisher, B. Y. Karlan, M. K. Agarwal, J. Higgins, C. Friedman, E. Villegas,C. Jacquemont, D. J. Farrugia, F. J. Couch, N. Urban, T. Taniguchi, Secondary mutations asamechanism of cisplatin resistance in BRCA2-mutated cancers. Nature451, 1116–1120 (2008).

25. S. J. Pettitt, F. L. Rehman, I. Bajrami, R. Brough, F. Wallberg, I. Kozarewa, K. Fenwick,I. Assiotis, L. Chen, J. Campbell, C. J. Lord, A. Ashworth, A genetic screen using the PiggyBactransposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity. PLOS ONE8, e61520 (2013).

26. S. F. Bunting, E. Callén, N. Wong, H.-T. Chen, F. Polato, A. Gunn, A. Bothmer, N. Feldhahn,O. Fernandez-Capetillo, L. Cao, X. Xu, C.-X. Deng, T. Finkel, M. Nussenzweig, J. M. Stark,A. Nussenzweig, 53BP1 inhibits homologous recombination in Brca1-deficient cellsby blocking resection of DNA breaks. Cell 141, 243–254 (2010).

27. J. E. Jaspers, A. Kersbergen, U. Boon, W. Sol, L. van Deemter, S. A. Zander, R. Drost,E. Wientjens, J. Ji, A. Aly, J. H. Doroshow, A. Cranston, N. M. B. Martin, A. Lau, M. J. O’Connor,S. Ganesan, P. Borst, J. Jonkers, S. Rottenberg, Loss of 53BP1 causes PARP inhibitorresistance in Brca1-mutated mouse mammary tumors. Cancer Discov. 3, 68–81 (2013).

10 of 12

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

by guest on October 3, 2020

http://stm.sciencem

ag.org/D

ownloaded from

28. G. Xu, J. R. Chapman, I. Brandsma, J. Yuan, M. Mistrik, P. Bouwman, J. Bartkova, E. Gogola,D. Warmerdam, M. Barazas, J. E. Jaspers, K. Watanabe, M. Pieterse, A. Kersbergen,W. Sol, P. H. N. Celie, P. C. Schouten, B. van den Broek, A. Salman, M. Nieuwland, I. de Rink,J. de Ronde, K. Jalink, S. J. Boulton, J. Chen, D. C. van Gent, J. Bartek, J. Jonkers,P. Borst, S. Rottenberg, REV7 counteracts DNA double-strand break resection and affectsPARP inhibition. Nature 521, 541–544 (2015).

29. A. Ray Chaudhuri, E. Callen, X. Ding, E. Gogola, A. A. Duarte, J.-E. Lee, N. Wong, V. Lafarga,J. A. Calvo, N. J. Panzarino, S. John, A. Day, A. V. Crespo, B. Shen, L. M. Starnes,J. R. de Ruiter, J. A. Daniel, P. A. Konstantinopoulos, D. Cortez, S. B. Cantor,O. Fernandez-Capetillo, K. Ge, J. Jonkers, S. Rottenberg, S. K. Sharan, A. Nussenzweig,Replication fork stability confers chemoresistance in BRCA-deficient cells.Nature 535, 382–387 (2016).

30. N. Johnson, Y.-C. Li, Z. E. Walton, K. A. Cheng, D. Li, S. J. Rodig, L. A. Moreau, C. Unitt,R. T. Bronson, H. D. Thomas, D. R. Newell, A. D. D’Andrea, N. J. Curtin, K.-K. Wong,G. I. Shapiro, Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARPinhibition. Nat. Med. 17, 875–882 (2011).

31. Y. H. Ibrahim, C. García-García, V. Serra, L. He, K. Torres-Lockhart, A. Prat, P. Anton,P. Cozar, M. Guzmán, J. Grueso, O. Rodríguez, M. T. Calvo, C. Aura, O. Díez, I. T. Rubio,J. Pérez, J. Rodón, J. Cortés, L. W. Ellisen, M. Scaltriti, J. Baselga, PI3K inhibition impairsBRCA1/2 expression and sensitizes BRCA-proficient triple-negative breast cancer to PARPinhibition. Cancer Discov. 2, 1036–1047 (2012).

32. A. Juvekar, L. N. Burga, H. Hu, E. P. Lunsford, Y. H. Ibrahim, J. Balmaña, A. Rajendran, A. Papa,K. Spencer, C. A. Lyssiotis, C. Nardella, P. P. Pandolfi, J. Baselga, R. Scully, J. M. Asara,L. C. Cantley, G. M. Wulf, Combining a PI3K inhibitor with a PARP inhibitor provides aneffective therapy for BRCA1-related breast cancer. Cancer Discov. 2, 1048–1063 (2012).

33. I. Bajrami, A. Kigozi, A. Van Weverwijk, R. Brough, J. Frankum, C. J. Lord, A. Ashworth,Synthetic lethality of PARP and NAMPT inhibition in triple-negative breast cancer cells.EMBO Mol. Med. 4, 1087–1096 (2012).

34. J. F. Liu, W. T. Barry, M. Birrer, J.-M. Lee, R. J. Buckanovich, G. F. Fleming, B. J. Rimel,M. K. Buss, S. Nattam, J. Hurteau, W. Luo, P. Quy, C. Whalen, L. Obermayer, H. Lee,E. P. Winer, E. C. Kohn, S. P. Ivy, U. A. Matulonis, Combination cediranib and olaparibversus olaparib alone for women with recurrent platinum-sensitive ovarian cancer:A randomised phase 2 study. Lancet Oncol. 15, 1207–1214 (2014).

35. K. Ha, W. Fiskus, D. S. Choi, S. Bhaskara, L. Cerchietti, S. G. T. Devaraj, B. Shah,S. Sharma, J. C. Chang, A. M. Melnick, S. Hiebert, K. N. Bhalla, Histone deacetylase inhibitortreatment induces ‘BRCAness’ and synergistic lethality with PARP inhibitor and cisplatinagainst human triple negative breast cancer cells. Oncotarget 5, 5637–5650 (2014).

36. Y. Du, H. Yamaguchi, Y. Wei, J. L. Hsu, H.-L. Wang, Y.-H. Hsu, W.-C. Lin, W.-H. Yu,P. G. Leonard, G. R. Lee IV, M.-K. Chen, K. Nakai, M.-C. Hsu, C.-T. Chen, Y. Sun, Y. Wu,W.-C. Chang, W.-C. Huang, C.-L. Liu, Y.-C. Chang, C.-H. Chen, M. Park, P. Jones,G. N. Hortobagyi, M.-C. Hung, Blocking c-Met–mediated PARP1 phosphorylationenhances anti-tumor effects of PARP inhibitors. Nat. Med. 22, 194–201 (2016).

37. W. Jdey, S. Thierry, C. Russo, F. Devun, M. Al Abo, P. Noguiez-Hellin, J.-S. Sun, E. Barillot,A. Zinovyev, I. Kuperstein, Y. Pommier, M. Dutreix, Drug-driven synthetic lethality:Bypassing tumor cell genetics with a combination of AsiDNA and PARP inhibitors. Clin.Cancer Res. 23, 1001–1011 (2017).

38. S. F. Johnson, C. Cruz, A. K. Greifenberg, S. Dust, D. G. Stover, D. Chi, B. Primack,S. Cao, A. J. Bernhardy, R. Coulson, J.-B. Lazaro, B. Kochupurakkal, H. Sun, C. Unitt,L. A. Moreau, K. A. Sarosiek, M. Scaltriti, D. Juric, J. Baselga, A. L. Richardson, S. J. Rodig,A. D. D’Andrea, J. Balmaña, N. Johnson, M. Geyer, V. Serra, E. Lim, G. I. Shapiro, CDK12inhibition reverses de novo and acquired PARP inhibitor resistance in BRCA wild-type andmutated models of triple-negative breast cancer. Cell Rep. 17, 2367–2381 (2016).

39. C. Sun, Y. Fang, J. Yin, J. Chen, Z. Ju, D. Zhang, X. Chen, C. P. Vellano, K. J. Jeong,P. K.-S. Ng, A. K. B. Eterovic, N. H. Bhola, Y. Lu, S. N. Westin, J. R. Grandis, S.-Y. Lin, K. L. Scott,G. Peng, J. Brugge, G. B. Mills, Rational combination therapy with PARP and MEKinhibitors capitalizes on therapeutic liabilities in RAS mutant cancers.Sci. Transl. Med. 9, eaal5148 (2017).

40. S. Shu, K. Polyak, BET bromodomain proteins as cancer therapeutic targets. Cold SpringHarb. Symp. Quant. Biol. 81, 1–9 (2017).

41. J. Shi, C. R. Vakoc, The mechanisms behind the therapeutic activity of BET bromodomaininhibition. Mol. Cell 54, 728–736 (2014).

42. M. A. Dawson, R. K. Prinjha, A. Dittmann, G. Giotopoulos, M. Bantscheff, W.-I. Chan,S. C. Robson, C.-w. Chung, C. Hopf, M. M. Savitski, C. Huthmacher, E. Gudgin, D. Lugo,S. Beinke, T. D. Chapman, E. J. Roberts, P. E. Soden, K. R. Auger, O. Mirguet, K. Doehner,R. Delwel, A. K. Burnett, P. Jeffrey, G. Drewes, K. Lee, B. J. P. Huntly, T. Kouzarides,Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusionleukaemia. Nature 478, 529–533 (2011).

43. J. E. Delmore, G. C. Issa, M. E. Lemieux, P. B. Rahl, J. W. Shi, H. M. Jacobs, E. Kastritis,T. Gilpatrick, R. M. Paranal, J. Qi, M. Chesi, A. C. Schinzel, M. R. McKeown, T. P. Heffernan,C. R. Vakoc, P. L. Bergsagel, I. M. Ghobrial, P. G. Richardson, R. A. Young, W. C. Hahn,K. C. Anderson, A. L. Kung, J. E. Bradner, C. S. Mitsiades, BET bromodomain inhibition as atherapeutic strategy to target c-Myc. Cell 146, 904–917 (2011).

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

44. J. A. Mertz, A. R. Conery, B. M. Bryant, P. Sandy, S. Balasubramanian, D. A. Mele,L. Bergeron, R. J. Sims III, Targeting MYC dependence in cancer by inhibiting BETbromodomains. Proc. Natl. Acad. Sci. U.S.A. 108, 16669–16674 (2011).

45. J. Zuber, J. Shi, E. Wang, A. R. Rappaport, H. Herrmann, E. A. Sison, D. Magoon,J. Qi, K. Blatt, M. Wunderlich, M. J. Taylor, C. Johns, A. Chicas, J. C. Mulloy, S. C. Kogan,P. Brown, P. Valent, J. E. Bradner, S. W. Lowe, C. R. Vakoc, RNAi screen identifies Brd4 as atherapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011).

46. W. W. Lockwood, K. Zejnullahu, J. E. Bradner, H. Varmus, Sensitivity of human lungadenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins.Proc. Natl. Acad. Sci. U.S.A. 109, 19408–19413 (2012).

47. A. Puissant, S. M. Frumm, G. Alexe, C. F. Bassil, J. Qi, Y. H. Chanthery, E. A. Nekritz, R. Zeid,W. C. Gustafson, P. Greninger, M. J. Garnett, U. McDermott, C. H. Benes, A. L. Kung,W. A. Weiss, J. E. Bradner, K. Stegmaier, Targeting MYCN in neuroblastoma by BETbromodomain inhibition. Cancer Discov. 3, 308–323 (2013).

48. J. Lovén, H. A. Hoke, C. Y. Lin, A. Lau, D. A. Orlando, C. R. Vakoc, J. E. Bradner,T. I. Lee, R. A. Young, Selective inhibition of tumor oncogenes by disruption ofsuper-enhancers. Cell 153, 320–334 (2013).

49. B. Chapuy, M. R. McKeown, C. Y. Lin, S. Monti, M. G. M. Roemer, J. Qi, P. B. Rahl, H. H. Sun,K. T. Yeda, J. G. Doench, E. Reichert, A. L. Kung, S. J. Rodig, R. A. Young, M. A. Shipp,J. E. Bradner, Discovery and characterization of super-enhancer-associated dependenciesin diffuse large B cell lymphoma. Cancer Cell 24, 777–790 (2013).

50. I. A. Asangani, V. L. Dommeti, X. Wang, R. Malik, M. Cieslik, R. Yang, J. Escara-Wilke,K. Wilder-Romans, S. Dhanireddy, C. Engelke, M. K. Iyer, X. Jing, Y.-M. Wu, X. Cao, Z. S. Qin,S. Wang, F. Y. Feng, A. M. Chinnaiyan, Therapeutic targeting of BET bromodomainproteins in castration-resistant prostate cancer. Nature 510, 278–282 (2014).

51. M. G. Baratta, A. C. Schinzel, Y. Zwang, P. Bandopadhayay, C. Bowman-Colin, J. Kutt,J. Curtis, H. Piao, L. C. Wong, A. L. Kung, R. Beroukhim, J. E. Bradner, R. Drapkin, W. C. Hahn,J. F. Liu, D. M. Livingston, An in-tumor genetic screen reveals that the BETbromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma.Proc. Natl. Acad. Sci. U.S.A. 112, 232–237 (2015).

52. S. Shu, C. Y. Lin, H. H. He, R. M. Witwicki, D. P. Tabassum, J. M. Roberts, M. Janiszewska,S. J. Huh, Y. Liang, J. Ryan, E. Doherty, H. Mohammed, H. Guo, D. G. Stover,M. B. Ekram, G. Peluffo, J. Brown, C. D’Santos, I. E. Krop, D. Dillon, M. McKeown, C. Ott,J. Qi, M. Ni, P. K. Rao, M. Duarte, S.-Y. Wu, C.-M. Chiang, L. Anders, R. A. Young,E. P. Winer, A. Letai, W. T. Barry, J. S. Carroll, H. W. Long, M. Brown, X. S. Liu, C. A. Meyer,J. E. Bradner, K. Polyak, Response and resistance to BET bromodomain inhibitors intriple-negative breast cancer. Nature 529, 413–414 (2016).

53. P. K. Mazur, A. Herner, S. S. Mello, M. Wirth, S. Hausmann, F. J. Sánchez-Rivera,S. M. Lofgren, T. Kuschma, S. A. Hahn, D. Vangala, M. Trajkovic-Arsic, A. Gupta, I. Heid,P. B. Noël, R. Braren, M. Erkan, J. Kleeff, B. Sipos, L. C. Sayles, M. Heikenwalder, E. Heßmann,V. Ellenrieder, I. Esposito, T. Jacks, J. E. Bradner, P. Khatri, E. A. Sweet-Cordero, L. D. Attardi,R. M. Schmid, G. Schneider, J. Sage, J. T. Siveke, Combined inhibition of BET familyproteins and histone deacetylases as a potential epigenetics-based therapy for pancreaticductal adenocarcinoma. Nat. Med. 21, 1163–1171 (2015).

54. N. Tasdemir, A. Banito, J.-S. Roe, D. Alonso-Curbelo, M. Camiolo, D. F. Tschaharganeh,C.-H. Huang, O. Aksoy, J. E. Bolden, C.-C. Chen, M. Fennell, V. Thapar, A. Chicas, C. R. Vakoc,S. W. Lowe, BRD4 connects enhancer remodeling to senescence immune surveillance.Cancer Discov. 6, 612–629 (2016).

55. A. Stathis, E. Zucca, M. Bekradda, C. Gomez-Roca, J.-P. Delord, T. de La Motte Rouge,E. Uro-Coste, F. de Braud, G. Pelosi, C. A. French, Clinical response of carcinomas harboringthe BRD4–NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628.Cancer Discov. 6, 492–500 (2016).

56. C. Berthon, E. Raffoux, X. Thomas, N. Vey, C. Gomez-Roca, K. Yee, D. C. Taussig, K. Rezai,C. Roumier, P. Herait, C. Kahatt, B. Quesnel, M. Michallet, C. Recher, F. Lokiec,C. Preudhomme, H. Dombret, Bromodomain inhibitor OTX015 in patients withacute leukaemia: A dose-escalation, phase 1 study. Lancet Haematol. 3, e186–e195(2016).

57. S. Amorim, A. Stathis, M. Gleeson, S. Iyengar, V. Magarotto, X. Leleu, F. Morschhauser,L. Karlin, F. Broussais, K. Rezai, P. Herait, C. Kahatt, F. Lokiec, G. Salles, T. Facon, A. Palumbo,D. Cunningham, E. Zucca, C. Thieblemont, Bromodomain inhibitor OTX015 in patientswith lymphoma or multiple myeloma: A dose-escalation, open-label, pharmacokinetic,phase 1 study. Lancet Haematol. 3, e196–e204 (2016).

58. Y. Yokoyama, H. Zhu, J. H. Lee, A. V. Kossenkov, S. Y. Wu, J. M. Wickramasinghe, X. Yin,K. C. Palozola, A. Gardini, L. C. Showe, K. S. Zaret, Q. Liu, D. Speicher, J. R. Conejo-Garcia,J. E. Bradner, Z. Zhang, A. K. Sood, T. Ordog, B. G. Bitler, R. Zhang, BET inhibitorssuppress ALDH activity by targeting ALDH1A1 super-enhancer in ovarian cancer. CancerRes. 76, 6320–6330 (2016).

59. Z. Mao, A. Seluanov, Y. Jiang, V. Gorbunova, TRF2 is required for repair of nontelomericDNA double-strand breaks by homologous recombination. Proc. Natl. Acad. Sci. U.S.A.104, 13068–13073 (2007).

60. B. P. Schlegel, F. M. Jodelka, R. Nunez, BRCA1 promotes induction of ssDNA by ionizingradiation. Cancer Res. 66, 5181–5189 (2006).

11 of 12

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

61. S. R. Floyd, M. E. Pacold, Q. Huang, S. M. Clarke, F. C. Lam, I. G. Cannell, B. D. Bryson,J. Rameseder, M. J. Lee, E. J. Blake, A. Fydrych, R. Ho, B. A. Greenberger, G. C. Chen, A. Maffa,A. M. Del Rosario, D. E. Root, A. E. Carpenter, W. C. Hahn, D. M. Sabatini, C. C. Chen,F. M. White, J. E. Bradner, M. B. Yaffe, The bromodomain protein Brd4 insulates chromatinfrom DNA damage signalling. Nature 498, 246–250 (2013).

Acknowledgments: We thank J. E. Bradner and J. Qi (Harvard Medical School) for providingJQ1. Funding: This work was supported, in whole or in part, by the Basser Center for BRCA(to L.Z.), the Harry Fields Professorship (to L.Z.), the NIH (R01CA142776, R01CA190415,P50CA083638, and P50CA174523 to L.Z., R01CA148759, R21CA198558, and U01CA200495 toQ.H., and R01NS094533 to Y.F.), the Breast Cancer Alliance (to L.Z. and C.V.D.), the OvarianCancer Research Fund (to X.H. and L.Z.), the Foundation for Women’s Cancer (to X.H. and L.Z.),the Kaleidoscope of Hope Ovarian Cancer Foundation (to L.Z.), and the Marsha RivkinCenter for Ovarian Cancer Research (to L.Z.). L.Y. and W.S. were supported by the ChinaScholarship Council. Author contributions: L.Y. and Y.Z. performed the experiments,analyzed the data, and wrote the article. W.S., L.F., Z.T., C.L., J.P., Y.W., and X.H. performed the

Yang et al., Sci. Transl. Med. 9, eaal1645 (2017) 26 July 2017

experiments and analyzed the data. Z.H. and J.Y. performed bioinformatics analyses.J.L.T. and K.M. reviewed the clinical and pathological data of TCGA. Q.H. and Y.F. providedexperimental materials and cell-based models. C.V.D. and L.Z. designed the experimentsand wrote the article. Competing interests: The authors declare that they have nocompeting interests.

Submitted 9 October 2016Resubmitted 12 April 2017Accepted 5 July 2017Published 26 July 201710.1126/scitranslmed.aal1645

Citation: L. Yang, Y. Zhang, W. Shan, Z. Hu, J. Yuan, J. Pi, Y. Wang, L. Fan, Z. Tang, C. Li, X. Hu,J. L. Tanyi, Y. Fan, Q. Huang, K. Montone, C. V. Dang, L. Zhang, Repression of BET activitysensitizes homologous recombination–proficient cancers to PARP inhibition. Sci. Transl. Med.9, eaal1645 (2017).

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PARP inhibitionproficient cancers to−Repression of BET activity sensitizes homologous recombination

ZhangTang, Chunsheng Li, Xiaowen Hu, Janos L. Tanyi, Yi Fan, Qihong Huang, Kathleen Montone, Chi V. Dang and Lin Lu Yang, Youyou Zhang, Weiwei Shan, Zhongyi Hu, Jiao Yuan, Jingjiang Pi, Yueying Wang, Lingling Fan, Zhaoqing

DOI: 10.1126/scitranslmed.aal1645, eaal1645.9Sci Transl Med

target many other tumor types, suggesting that this combination treatment may have broad applicability.models of breast and ovarian cancer. They also provided genetic data showing that BET inhibitors may be able to sensitizes tumors to PARP inhibitors. The authors demonstrated the effectiveness of this combination approach inThe authors found that BET inhibitors suppress homologous recombination, inducing the functional defect that

inhibitors.screen and discovered that BET inhibitors, a type of epigenetic regulators, act synergistically with PARP . performed a druget alrecombination. To identify drugs that may potentiate the activity of PARP inhibitors, Yang

PARP inhibitors are drugs that interfere with DNA repair, particularly in cancers with defects in homologousBET-ting on a new drug combination

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