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MicroRNA (miRNA) dysregulation is a hallmark of cutaneous T-cell lymphoma (CTCL), an often-fatal malignancy of skin-homing CD4+ T cells for which there are few effective therapies. The role of microRNAs (miRs) in controlling epigenetic modifier-dependent transcriptional regulation in CTCL is unknown. In this study, we characterize a novel miR dysregulation that contributes to overexpression of the epigenetic reader bromodomain-containing protein 4 (BRD4). We used patient CD4+ T cells to show diminished levels of miR-29b compared with healthy donor cells. Patient cells and miR-29b-/- mouse cells revealed an inverse relationship between miR-29b and BRD4, the latter of which is overexpressed in these cells. Chromatin immunoprecipitation and sequencing analysis revealed increased genome-wide BRD4 occupancy at promoter and enhancer regions in CD4+ T cells from CTCL patients. The cumulative result of BRD4 binding was increased expression of tumor-associated genes such as NOTCH1 and RBPJ, as well as the interleukin-15 (IL-15) receptor complex, the latter enhancing IL-15 autocrine signaling. Furthermore, we confirm the in vivo relevance of this pathway in our IL-15 transgenic mouse model of CTCL by showing that interference with BRD4-mediated pathogenesis, either by restoring miR-29b levels via bortezomib treatment or by directly inhibiting BRD4 binding via JQ1 treatment, prevents progression of CTCL. We describe a novel oncogenic pathway featuring IL-15, miR-29b, and BRD4 in CTCL and suggest targeting of these components as a potentially effective therapy for CTCL patients.

SUMMARY Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

While combinatory antiretroviral therapy (cART) can effectively reduce HIV-1 viremia, it cannot eliminate HIV-1 infection. In the presence of cART, viral reservoirs remain latent, impeding the cure of HIV-1/AIDS. Recently, latency-reversing agents (LRAs) have been developed with the intent of purging latent HIV-1, providing an intriguing strategy for the eradication of the residual viral reservoirs. Our earlier studies show that the first-generation, methyl-triazolo bromodomain, and extra-terminal domain inhibitor (BETi), JQ1, facilitates the reversal of HIV-1 latency. BETis have emerged as a new class of compounds that are promising for this HIV-1 “shock and kill” eradication approach. However, when used as a single drug, JQ1 only modestly reverses HIV-1 latency, which complicates studying the underlining mechanisms. Meanwhile, it has been widely discussed that the induction of latent proviruses is stochastic (Ho et al., 2013). Thus, new BETis are currently under active development with focus on improving potency, ease of synthesis and structural diversity. Using fluorous-tagged multicomponent reactions, we developed a novel second-generation, 3,5-dimethylisoxazole BETi based on an imidazo[1,2-a] pyrazine scaffold, UMB-32. Furthermore, we screened 37 UMB-32 derivatives and identified that one, UMB-136, reactivates HIV-1 in multiple cell models of HIV-1 latency with better efficiency than either JQ1 or UMB-32. UMB-136 enhances HIV-1 transcription and increases viral production through the release of P-TEFb. Importantly, UMB-136 enhances the latency-reversing effects of PKC agonists (prostratin, bryostatin-1) in CD8-depleted PBMCs containing latent viral reservoirs. Our results illustrate that structurally improved BETis, such as UMB-136, may be useful as promising LRAs for HIV-1 eradication.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

Despite current standard of care, the average 5-year mortality after an initial diagnosis of heart failure (HF) is about 40%, reflecting an urgent need for new therapeutic approaches. Previous studies demonstrated that the epigenetic reader protein bromodomain-containing protein 4 (BRD4), an emerging therapeutic target in cancer, functions as a critical coactivator of pathologic gene transactivation during cardiomyocyte hypertrophy. However, the therapeutic relevance of these findings to human disease remained unknown. We demonstrate that treatment with the BET bromodomain inhibitor JQ1 has therapeutic effects during severe, preestablished HF from prolonged pressure overload, as well as after a massive anterior myocardial infarction in mice. Furthermore, JQ1 potently blocks agonist-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Integrated transcriptomic analyses across animal models and human iPSC-CMs reveal that BET inhibition preferentially blocks transactivation of a common pathologic gene regulatory program that is robustly enriched for NFκB and TGF-β signaling networks, typified by innate inflammatory and profibrotic myocardial genes. As predicted by these specific transcriptional mechanisms, we found that JQ1 does not suppress physiological cardiac hypertrophy in a mouse swimming model. These findings establish that pharmacologically targeting innate inflammatory and profibrotic myocardial signaling networks at the level of chromatin is effective in animal models and human cardiomyocytes, providing the critical rationale for further development of BET inhibitors and other epigenomic medicines for HF.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N

Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4. Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

Cancer cell hallmarks are underpinned by transcriptional programmes operating in the context of a dynamic and complicit epigenomic environment. Somatic alterations of chromatin modifiers are among the most prevalent cancer perturbations. There is a pressing need for targeted chemical probes to dissect these complex, interconnected gene regulatory circuits. Validated chemical probes empower mechanistic research while providing the pharmacological proof of concept that is required to translate drug-like derivatives into therapy for cancer patients. In this Review, we describe chemical probe development for epigenomic effector proteins that are linked to cancer pathogenesis. By annotating these reagents, we aim to share our perspectives on an informative 'epigenomic toolbox' of broad utility to the research community.

// Montserrat Perez-Salvia 1 , Laia Simo-Riudalbas 1 , Pere Llinas-Arias 1 , Laura Roa 1 , Fernando Setien 1 , Marta Soler 1 , Manuel Castro de Moura 1 , James E. Bradner 2 , Eva Gonzalez-Suarez 1 , Catia Moutinho 1 and Manel Esteller 1, 3, 4 1 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain 2 Novartis Institutes for Biomedical Research, Cambridge, MA, USA 3 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, Spain 4 Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Catalonia, Spain Correspondence to: Catia Moutinho, email: cmoutinho@idibell.cat Manel Esteller, email: mesteller@idibell.cat Keywords: luminal breast cancer, bromodomain inhibitor, C-MYC, JQ1, mice model Received: February 28, 2017 Accepted: May 04, 2017 Published: May 29, 2017 ABSTRACT BET bromodomain inhibitors, which have an antitumoral effect against various solid cancer tumor types, have not been studied in detail in luminal breast cancer, despite the prevalence of this subtype of mammary malignancy. Here we demonstrate that the BET bromodomain inhibitor JQ1 exerts growth-inhibitory activity in human luminal breast cancer cell lines associated with a depletion of the C-MYC oncogene, but does not alter the expression levels of the BRD4 bromodomain protein. Interestingly, expression microarray analyses indicate that, upon JQ1 administration, the antitumoral phenotype also involves downregulation of relevant breast cancer oncogenes such as the Breast Carcinoma-Amplified Sequence 1 (BCAS1) and the PDZ Domain-Containing 1 (PDZK1). We have also applied these in vitro findings in an in vivo model by studying a transgenic mouse model representing the luminal B subtype of breast cancer, the MMTV-PyMT, in which the mouse mammary tumor virus promoter is used to drive the expression of the polyoma virus middle T-antigen to the mammary gland. We have observed that the use of the BET bromodomain inhibitor for the treatment of established breast neoplasms developed in the MMTV-PyMT model shows antitumor potential. Most importantly, if JQ1 is given before the expected time of tumor detection in the MMTV-PyMT mice, it retards the onset of the disease and increases the survival of these animals. Thus, our findings indicate that the use of bromodomain inhibitors is of great potential in the treatment of luminal breast cancer and merits further investigation.

Cancer arises from genetic alterations that invariably lead to dysregulated transcriptional programs. These dysregulated programs can cause cancer cells to become highly dependent on certain regulators of gene expression. Here, we discuss how transcriptional control is disrupted by genetic alterations in cancer cells, why transcriptional dependencies can develop as a consequence of dysregulated programs, and how these dependencies provide opportunities for novel therapeutic interventions in cancer., National Institutes of Health (U.S.) (Grant HG002668)

In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival., Active enhancers are defined by the presence of post-translational modifications of histones. Here, the authors use these marks to identify enhancers recurrently activated in colorectal cancer and find that these enhancers turn on oncogenes and are associated with known risk loci for developing the disease.

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1-9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

Summary Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment., Graphical Abstract, Highlights • vHMECs arise through epigenetic modification of pre-existing human breast cells • DNA methylation by DNMT3a is required for vHMEC formation • Loss of DOT1L and active histone methylation marks accelerates vHMEC formation • Loss of mammary identity requires changes in both DNA and histone methylation, In this article, Kuperwasser and colleagues show that, upon removal from their native microenvironment, rare pre-existing human mammary epithelial cells undergo stochastic epigenetic reprogramming and lose mammary lineage identity. This dedifferentiation occurs through addition of repressive promoter methylation marks by DNMT3a and removal of active histone methylation marks through DOT1L loss, which together suppress mammary lineage commitment gene expression.

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1–S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2–M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR–Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. Cancer Res; 74(24); 7534–45. ©2014 AACR.

SummaryActivation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.

SummaryEpigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is a prevailing methodology used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx) that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.

Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mammalian target of rapamycin (mTOR) inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of bromodomain-containing protein 4 (BRD4) inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains the antiproliferative activity of JQ1 in OS cells.

BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors.

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34+ human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34+ normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI–resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD–expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI–resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI. Mol Cancer Ther; 13(10); 2315–27. ©2014 AACR.

Proinflammatory stimuli elicit rapid transcriptional responses via transduced signals to master regulatory transcription factors. To explore the role of chromatin-dependent signal transduction in the atherogenic inflammatory response, we characterized the dynamics, structure, and function of regulatory elements in the activated endothelial cell epigenome. Stimulation with tumor necrosis factor alpha prompted a dramatic and rapid global redistribution of chromatin activators to massive de novo clustered enhancer domains. Inflammatory super enhancers formed by nuclear factor-kappa B accumulate at the expense of immediately decommissioned, basal endothelial super enhancers, despite persistent histone hyperacetylation. Mass action of enhancer factor redistribution causes momentous swings in transcriptional initiation and elongation. A chemical genetic approach reveals a requirement for BET bromodomains in communicating enhancer remodeling to RNA Polymerase II and orchestrating the transition to the inflammatory cell state, demonstrated in activated endothelium and macrophages. BET bromodomain inhibition abrogates super enhancer-mediated inflammatory transcription, atherogenic endothelial responses, and atherosclerosis in vivo.

7,9-Diaryl-1,6,8-trioxaspiro[4.5]dec-3-en-2-ones are a recently described group of spirocyclic butenolides that can be generated rapidly and as a single diastereomer through a cascade process between γ-hydroxybutenolides and aromatic aldehydes. The following outlines our findings that these spirocycles are potently cytotoxic and have a dramatic structure–function profile that provides excellent insight into the structural features required for this potency.

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.

The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy.

The identification of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL) led to clinical testing of γ-secretase inhibitors (GSIs) that prevent NOTCH1 activation. However, responses to these inhibitors have been transient, suggesting that resistance limits their clinical efficacy. Here we modeled T-ALL resistance, identifying GSI-tolerant 'persister' cells that expand in the absence of NOTCH1 signaling. Rare persisters are already present in naive T-ALL populations, and the reversibility of their phenotype suggests an epigenetic mechanism. Relative to GSI-sensitive cells, persister cells activate distinct signaling and transcriptional programs and exhibit chromatin compaction. A knockdown screen identified chromatin regulators essential for persister viability, including BRD4. BRD4 binds enhancers near critical T-ALL genes, including MYC and BCL2. The BRD4 inhibitor JQ1 downregulates expression of these targets and induces growth arrest and apoptosis in persister cells, at doses well tolerated by GSI-sensitive cells. Consistently, the GSI-JQ1 combination was found to be effective against primary human leukemias in vivo. Our findings establish a role for epigenetic heterogeneity in leukemia resistance that may be addressed by incorporating epigenetic modulators in combination therapy.

Pioneering work over the past years has highlighted the remarkable ability of manipulating cell states through exogenous, mostly transcription factor-induced reprogramming. The use of small molecules and reprogramming by transcription factors share a common history starting with the early AZA and MyoD experiments in fibroblast cells. Recent work shows that a combination of small molecules can replace all of the reprogramming factors and many previous studies have demonstrated their use in enhancing efficiencies or replacing individual factors. Here we provide a brief introduction to reprogramming followed by a detailed review of the major classes of small molecules that have been used to date and what future opportunities can be expected from these.

Genetically engineered mouse (GEM) models are a pillar of functional cancer research. Here, we developed RapidCaP, a GEM modeling system that uses surgical injection for viral gene delivery to the prostate. We show that in Pten deficiency, loss of p53 suffices to trigger metastasis to distant sites at greater than 50% penetrance by four months, consistent with results from human prostate cancer genome analysis. Live bioluminescence tracking showed that endogenous primary and metastatic disease responds to castration before developing lethal castration resistance. To our surprise, the resulting lesions showed no activation of Akt but activation of the Myc oncogene. Using RapidCaP, we find that Myc drives local prostate metastasis and is critical for maintenance of metastasis, as shown by using the Brd4 inhibitor JQ1. Taken together, our data suggest that a “MYC-switch” away from AKT forms a critical and druggable event in PTEN-mutant prostate cancer metastasis and castration resistance. Significance: The RapidCaP system introduces fast and flexible genetics for functional analysis and therapy for endogenous metastatic prostate cancer. The approach introduces targeting of MYC as a critical strategy against PTEN-deficient lethal prostate cancer. Cancer Discov; 4(3); 318–33. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 259

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Drug With a (Re)Purpose Thalidomide, once infamous for its deleterious effects on fetal development, has re-emerged as a drug of great interest because of its beneficial immunomodulatory effects. A derivative drug called lenalidomide significantly extends the survival of patients with multiple myeloma, but the molecular mechanisms underlying its efficacy remain unclear (see the Perspective by Stewart ). Building on a previous observation that thalidomide binds to cereblon, a ubiquitin ligase, Lu et al. (p. 305 , published online 28 November) and Krönke et al. (p. 301 , published online 28 November) show that in the presence of lenalidomide, cereblon selectively targets two B cell transcription factors (Ikaros family members, IKZF1 and IKZF3) for degradation. In myeloma cell lines and patient cells, down-regulation of IKZF1 and IKZF3 was necessary and sufficient for the drug's anticancer activity. Thus, lenalidomide may act, at least in part, by “grepurposing” a ubiquitin ligase.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Currently, there is a growing need for culturing hematopoietic stem/progenitor cells (HSPCs) in vitro for various clinical applications including gene therapy. Compared with cord blood (CB) CD34+ HSPCs, it is more challenging to maintain or expand CD34+ peripheral blood mobilized stem/progenitor cells (PBSCs) ex vivo. To fill this knowledge gap, we have systematically surveyed 466 small-molecule drug compounds for their potential in cytokine-dependent expansion of human CD34+CD90+ HSPCs. We found that epigenetic modifiers, especially histone deacetylase inhibitors (HDACis), could preferentially maintain and expand these cells. In particular, treatment of CD34+ PBSCs with a single dose of HDACi trichostatin A (TSA) at a concentration of 50 nmol/L ex vivo yielded the greatest expansion (11.7-fold) of CD34+CD90+ cells when compared with the control (dimethyl sulfoxide [DMSO] plus cytokines) group. Additionally, TSA-treated PBSC CD34+ cells had a statistically significant higher engraftment rate than the control-treated group in xenotransplantation experiments. Mechanistically, TSA treatment was associated with increased expression of HSPC-related genes such as GATA2 and SALL4. Furthermore, TSA-mediated CD34+CD90+ expansion was reduced by downregulation of SALL4 but not GATA2. Overall, we have developed a robust, short-term (5-day), PBSC ex vivo maintenance/expansion culture technique and found that the HDACi–TSA/SALL4 axis is important for the biological process.

A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide new and important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods provide a powerful approach to enhance understanding of therapeutic action and characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.

Purpose: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non–small cell lung cancer (NSCLC) with BET inhibition. Experimental Design: We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results: Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion: Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183–92. ©2013 AACR.

Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure.

Heart failure (HF) is driven by the interplay between master regulatory transcription factors and dynamic alterations in chromatin structure. While pathologic gene transactivation in this context is known to be associated with recruitment of histone acetyl-transferases and local chromatin hyperacetylation, the role of epigenetic reader proteins in cardiac biology is unknown. We therefore undertook a first study of acetyl-lysine reader proteins, or bromodomains, in HF. Using a chemical genetic approach, we establish a central role for BET-family bromodomain proteins in gene control during HF pathogenesis. BET inhibition potently suppresses cardiomyocyte hypertrophy in vitro and pathologic cardiac remodeling in vivo. Integrative transcriptional and epigenomic analyses reveal that BET proteins function mechanistically as pause-release factors critical to activation of canonical master regulators and effectors that are central to HF pathogenesis and relevant to the pathobiology of failing human hearts. This study implicates epigenetic readers in cardiac biology and identifies BET co-activator proteins as therapeutic targets in HF.

James E Bradner is an Assistant Professor in Medicine at Harvard Medical School (MA, USA) as well as a Staff Physician in the Division of Hematologic Malignancies at Dana-Farber Cancer Institute (MA, USA). The present research focus of the Bradner laboratory concerns the discovery and optimization of prototype drugs targeting cancer gene regulation. The clinical objective of the Bradner group is to deliver novel therapeutics for human clinical investigation in hematologic diseases. Bradner’s awards and honors include the Damon Runyon-Rachleff Innovation Award, the Smith Family Award for Excellence in Biomedical Research, the Dunkin’ Donuts Rising Star Award and the HMS Distinguished Excellence in Teaching Award. He is a member of the American Society of Clinical Investigation, the American Society of Hematology, the American Chemical Society and the American Association of Cancer Research. His recent research has been published in Nature, Cell, Nature Chemical Biology and the Journal of the American Chemical Society. He has authored more than 20 US Patent applications, licensed to five pharmaceutical companies, and is a scientific founder of Acetylon Pharmaceuticals, SHAPE Pharmaceuticals, Tensha Therapeutics and Syros Pharmaceuticals. Bradner received his AB from Harvard University, his MD from the University of Chicago (IL, USA) and a MMS from Harvard Medical School. He completed his postgraduate training in Internal Medicine at Brigham & Women’s Hospital (MA, USA), followed by a fellowship in Medical Oncology and Hematology at Dana-Farber Cancer Institute. Following additional post-doctoral training in Chemistry at Harvard University and the Broad Institute (MA, USA) with Professor Stuart Schreiber, Bradner joined the research faculty of Dana-Farber in 2008. Interview conducted by Hannah Coaker, Assistant Commissioning Editor.

Acetylation of the RelA subunit of NF-κB at lysine-310 regulates the transcriptional activation of NF-κB target genes and contributes to maintaining constitutively active NF-κB in tumors. Bromodomain-containing factor Brd4 has been shown to bind to acetylated lysine-310 (AcLys310) and to regulate the transcriptional activity of NF-κB, but the role of this binding in maintaining constitutively active NF-κB in tumors remains elusive. In this study, we demonstrate the structural basis for the binding of bromodomains (BDs) of bromodomain-containing protein 4 (Brd4) to AcLys310 and identify the BD inhibitor JQ1 as an effective small molecule to block this interaction. JQ1 suppresses TNF-α-mediated NF-κB activation and NF-κB-dependent target gene expression. In addition, JQ1 inhibits the proliferation and transformation potential of A549 lung cancer cells and suppresses the tumorigenicity of A549 cells in severe combined immunodeficiency mice. Furthermore, we demonstrate that depletion of Brd4 or treatment of cells with JQ1 induces the ubiquitination and degradation of the constitutively active nuclear form of RelA. Our results identify a novel function of Brd4 in maintaining the persistently active form of NF-κB found in tumors, and they suggest that interference with the interaction between acetylated RelA and Brd4 could be a potential therapeutic approach for the treatment of NF-κB-driven cancer.

SummaryWhile cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here, we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs shows that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is required to maintain MYC expression in NMC cell lines. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC.

Replication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase δ. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(+) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus.

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same exte...

A significant challenge and potential high-value application of computer-aided drug design is the accurate prediction of protein–ligand binding affinities. Free energy perturbation (FEP) using molecular dynamics (MD) sampling is among the most suitable approaches to achieve accurate binding free energy predictions, due to the rigorous statistical framework of the methodology, correct representation of the energetics, and thorough treatment of the important degrees of freedom in the system (including explicit waters). Recent advances in sampling methods and force fields coupled with vast increases in computational resources have made FEP a viable technology to drive hit-to-lead and lead optimization, allowing for more efficient cycles of medicinal chemistry and the possibility to explore much larger chemical spaces. However, previous FEP applications have focused on systems with high-resolution crystal structures of the target as starting points—something that is not always available in drug discovery projects. As such, the ability to apply FEP on homology models would greatly expand the domain of applicability of FEP in drug discovery. In this work we apply a particular implementation of FEP, called FEP+, on congeneric ligand series binding to four diverse targets: a kinase (Tyk2), an epigenetic bromodomain (BRD4), a transmembrane GPCR (A2A), and a protein–protein interaction interface (BCL-2 family protein MCL-1). We apply FEP+ using both crystal structures and homology models as starting points and find that the performance using homology models is generally on a par with the results when using crystal structures. The robustness of the calculations to structural variations in the input models can likely be attributed to the conformational sampling in the molecular dynamics simulations, which allows the modeled receptor to adapt to the “real” conformation for each ligand in the series. This work exemplifies the advantages of using all-atom simulation methods with full system flexibility and offers promise for the general application of FEP to homology models, although additional validation studies should be performed to further understand the limitations of the method and the scenarios where FEP will work best.

Inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) is the signature lesion in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). pVHL loss causes the transcriptional activation of hypoxia-inducible factor (HIF) target genes, including many genes that encode histone lysine demethylases. Moreover, chromatin regulators are frequently mutated in this disease. We found that ccRCC displays increased H3K27 acetylation and a shift toward mono- or unmethylated H3K27 caused by an HIF-dependent increase in H3K27 demethylase activity. Using a focused short hairpin RNA library, as well as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) and a pharmacological inhibitor, we discovered that pVHL-defective ccRCC cells are hyperdependent on the H3K27 methyltransferase EZH1 for survival. Therefore, targeting EZH1 could be therapeutically useful in ccRCC.

Bromodomain and extraterminal (BET) proteins are epigenetic “readers” that recognize acetylated histones and mark areas of the genome for transcription. BRD4, a BET family member protein, has been implicated in a number of types of cancer, and BET protein inhibitors (BETi) are efficacious in many preclinical cancer models. However, the drivers of response to BETi vary depending on tumor type, and little is known regarding the target genes conveying BETi activity in triple-negative breast cancer (TNBC). Here, we show that BETi repress growth of multiple in vitro and in vivo models of TNBC by inducing two terminal responses: apoptosis and senescence. Unlike in other cancers, response to BETi in TNBC is not dependent upon suppression of MYC. Instead, both end points are preceded by the appearance of polyploid cells caused by the suppression of Aurora kinases A and B (AURKA/B), which are critical mediators of mitosis. In addition, AURKA/B inhibitors phenocopy the effects of BETi. These results indicate that Aurora kinases play an important role in the growth suppressive activity of BETi in TNBC. Elucidating the mechanism of response to BETi in TNBC should 1) facilitate the prediction of how distinct TNBC tumors will respond to BETi and 2) inform the rational design of drug combination therapies.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Tyrosine kinase inhibitors (TKI) have revolutionized chronic myelogenous leukemia (CML) management. Disease eradication, however, is hampered by innate resistance of leukemia-initiating cells (LIC) to TKI-induced killing, which also provides the basis for subsequent emergence of TKI-resistant mutants. We report that EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is overexpressed in CML LICs and required for colony formation and survival and cell-cycle progression of CML cell lines. A critical role for EZH2 is supported by genetic studies in a mouse CML model. Inactivation of Ezh2 in conventional conditional mice and through CRISPR/Cas9-mediated gene editing prevents initiation and maintenance of disease and survival of LICs, irrespective of BCR–ABL1 mutational status, and extends survival. Expression of the EZH2 homolog EZH1 is reduced in EZH2-deficient CML LICs, creating a scenario resembling complete loss of PRC2. EZH2 dependence of CML LICs raises prospects for improved therapy of TKI-resistant CML and/or eradication of disease by addition of EZH2 inhibitors. Significance: This work defines EZH2 as a selective vulnerability for CML cells and their LICs, regardless of BCR–ABL1 mutational status. Our findings provide an experimental rationale for improving disease eradication through judicious use of EZH2 inhibitors within the context of standard-of-care TKI therapy. Cancer Discov; 6(11); 1237–47. ©2016 AACR. See related article by Scott et al., p. 1248. This article is highlighted in the In This Issue feature, p. 1197