ARTICLE RESEARCH cell nd analysis.Paired s and untreated animal prparestandardized protocols and commercially y ma ous differe el evant disease arkedt-tonductora iot in ds thor the tumo is by PET in aging (Fi (-JQ1 treatr d).Animals wererando vehicl 2h,as and al days of t uptake w vehie-re 5 of ac JQ1 (Supp mentary Fig.4c) aredforguantathreimmnohistoeh stry analysis demonstrate cas de BRD4 inhibi- ct o 1060 AN (r)( and late ar ed with ar nov and day ed,induces immediate and pr poptosis in BRD4 NMC Discussion (Supplementary Figs 8 and9) Across the complex landscape of the cancer genome,chromo cy of JQl in xenogr It models s of NMC enograft models st,short- rm treatment studie d info ailabl Herein,we pro id re rar sof the with JQl treatm vchicle-reated mice at this r weigh tary Fig.10b) mical probe 16 uclear Ki67 and ine ivatives for thera he ing drugp apeutic app -3 lemen cq 20 gh VEEMBE2010IVOL ATORE1071featuring cell spreading and flattening, open chromatin and striking spindle morphology (Fig. 4e and Supplementary Fig. 4). Differentiation is prompt (,24 h) and characterized by marked changes in cell shape accompanied by markedly augmented expression of cytokeratin, a hall￾mark of squamous differentiation (Fig. 4e). After 7 days in culture with submicromolar exposures to JQ1, terminal differentiation is observed. In this manner, JQ1 phenocopies the morphological changes and increased keratin expression observed with BRD4–NUT silencing by RNAinterference (Supplementary Fig. 5)17. Corroborating these studies, expression analysis of three canonical squamous tissue genes by RT– PCR identified marked (30-fold) induction of keratin-14 by (1)-JQ1 in NMC 797 cells (Fig. 4f). The modest induction of keratin-10 without affecting epidermal transglutaminase (TGM1) may indicate differenti￾ation towards thoracic squamous epithelium, consistent with the mediastinal primary tumour from which NMC 797 cells derive33. Induction of differentiation with intense keratin staining is progressive over 72 h, as determined by quantitative immunohistochemistry analysis (Supplementary Fig. 6). Supporting an on-target mechanism-of-action, the (2)-JQ1 enantiomer is comparatively inactive in NMC, and a non￾BRD4-dependent squamous carcinoma cell line (TE10) fails to exhibit differentiation effects of active JQ1 (Supplementary Fig. 4c). In BRD4-dependent NMC cells, differentiation is expectedly accompanied by growth arrest, as demonstrated by reduced Ki67 staining (Supplementary Fig. 7), sustained inhibition of proliferation (Fig. 4g; Supplementary Fig. 8) and G1 cell-cycle arrest (Fig. 4h). To understand further the observed G1 arrest and to confirm an effect of JQ1 on known BRD4-dependent genes, we performed quantitative RT–PCR for RAD21 and RAN (ref. 4). (1)-JQ1 potently decreased expression of both BRD4 target genes, whereas (2)-JQ1 had no effect (Fig. 4f). Early and late apoptosis were assessed with annexin-V and propidium iodide staining to ascertain whether the antiproliferative effect and irreversible differentiation was accompanied by cell death. Indeed, JQ1 induces immediate and progressive apoptosis in BRD4- dependent human carcinoma cells, without triggering significant growth arrest or cell death in cell lines lacking the BRD–NUT fusion (Supplementary Figs 8 and 9). Antitumour efficacy of JQ1 in xenograft models of NMC To determine whether JQ1 could attenuate the growth of BRD4- dependent carcinoma as a single agent in vivo, we developed three xenograft models of NMC in mice. First, short-term treatment studies were performed in NMC 797 xenografts with positron-emission tomography (PET) imaging of 18F-fluorodeoxyglucose (FDG) uptake as a primary endpoint to explore whether activity of JQ1 could be demonstrated by non-invasive imaging. Matched cohorts of mice with established tumours were randomized to treatment with JQ1 (50 mg kg21 ) or vehicle, administered by daily intraperitoneal injec￾tion. Before randomization, and after 4 days of therapy, mice were evaluated by FDG-PET imaging. A marked reduction in FDG uptake was observed with JQ1 treatment, whereas vehicle-treated mice demonstrated progressive disease (Fig. 5a). Tumour-volume measure￾ments confirmed a reduction in tumour growth with JQ1 treatment (Fig. 5b and Supplementary Fig. 10). JQ1 was well tolerated at this dose and schedule without overt signs of toxicity or weight loss (Supplemen￾tary Fig. 10b). To confirm that the antineoplastic effect observed with JQ1 treat￾ment was associated with target engagement, sectioned tumour tissue was examined for the BRD4–NUT oncoprotein. As presented in Sup￾plementary Fig. 11, JQ1 treatment resulted in effacement of NUT nuclear speckles, consistent with competitive binding to nuclear chromatin. Cell spreading and increased keratin expression confirmed induction of squamous differentiation (Fig. 5c). Decreased nuclearKi67 andincreased TUNEL staining in treated animals confirmed an ongoing antiprolifera￾tive, pro-apoptotic effect (Supplementary Fig. 11). To quantify the phar￾macodynamic biomarker of tumour keratin expression, we established protocols for automated immunohistochemistry image acquisition and analysis. Paired samples from treated and untreated animals were prepared and analysed using standardized protocols and commercially available software (ImageScope; Aperio Technologies), demonstrating that JQ1 induced strong (grade 31) keratin expression in NMC 797 xenografts (Supplementary Fig. 12). In parallel with these studies, we had occasion to care for a 29-year￾old patient with widely metastatic BRD4–NUT-positive NMC arising from the mediastinum. With the goal of developing a more clinically relevant disease model, we established short-term cultures (11060 cells) using discarded clinical material obtained from pleural fluid draining from a palliative chest tube. As presented in Supplemen￾tary Fig. 13, in vitro studies confirmed the stereospecific, potent effect of (1)-JQ1 on cellular viability (IC50 5 4 nM), growth and cell cycle progression. Four animals engrafted with patient-derived tumour material developed measurable disease, which was strongly FDG-avid by PET imaging (Fig. 5d). Animals were randomly assigned to vehicle or (1)-JQ1 treatment. Before treatment and after 4 days of therapy, mice were evaluated by PET imaging. A marked response in FDG uptake was observed with (1)-JQ1 treatment, whereas vehicle-treated animals demonstrated progressive disease (Fig. 5e). Tumour material preparedfor quantitative immunohistochemistry analysis demonstrated induction of keratin expression after (1)-JQ1 treatment (Fig. 5f, g and Supplementary Fig. 14) in this minimally passaged NMC xenograft model. To confirm the translational potential of direct-acting BRD4 inhibi￾tion in NMC, we further adapted the patient-derived 11060 cells to expansion in vivo, and performed definitive efficacy studies. Marked tumour regression and prolonged overall survival were observed, after only 18 days of well-tolerated therapy with (1)-JQ1 (Fig. 5h, i). These results were recapitulated in a third NMC xenograft model, using Per403 cells (Fig. 5j, k and Supplementary Fig. 15). Together, these data establish in vivo proof-of-concept for targeting BRD4 with JQ1 in NMC. Discussion Across the complex landscape of the cancer genome, recurrent chromo￾somal rearrangements comprise a compelling subset of clear, genetic targets in cancer. As evidenced by the successful development of first￾and second-generation kinase inhibitors targeting BCR–ABL in chronic myelogenous leukaemia, well-characterized probe compounds34,35, high-resolution crystallographic data36, translational research studies37, and informative murine models38, where available, provide an optimal platform for ligand discovery and target validation. Herein, we provide comparable evidence supporting the BRD4–NUT fusion as a thera￾peutic target in an incurable, genetically-defined human squamous carcinoma, using a novel BRD4-directed small-molecule inhibitor. Beyond NUT-midline carcinoma, BET-family bromodomains contribute to other neoplastic and non-neoplastic diseases. BRD4 targets the P-TEFb complex to mitotic chromosomes, resulting in the expression of growth-promoting genes such as c-Myc12,14 and the well-established cancer target Aurora B39. BET family members have been recognized as essential genesfor the replication of viruses40,41 and in mediating inflammatory responses42. Thus, the availability of (1)-JQ1 will prompt informative research broadly in developmental and disease biology. JQ1 possesses many desirable qualities of a chemical probe, such as high target potency in homogeneous and cellular assays, a well-characterized profile of selectivity, synthetic accessibility and herein proven utility in experimental biology18,19. We have also found JQ1 to exhibit few off-target effects on cellular receptors and excellent pharma￾cokinetic properties including 49% oral bioavailability (Supplementary Figs 16 and 17 and Supplementary Table 5), establishing the plausibility of developing drug-like derivatives for therapeutic application. The discovery and optimization of small-molecule inhibitors of epigenetic targets is a major focus of current biomedical research43. We sought to meet the challenge of developing potent, selective inhibitors of epigenetic readers. Here we present a first, thoroughly characterized ARTICLE RESEARCH 23/30 DECEMBER 2010 | VOL 468 | NATURE | 1071 ©2010 Macmillan Publishers Limited. All rights reserved