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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. [ABSTRACT FROM AUTHOR]

Summary: Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule “diversity microarrays” containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine. [Copyright &y& Elsevier]

Summary Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) “dropout screens” on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate “drivers,” and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors. [ABSTRACT FROM AUTHOR]

Summary We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core ( Exosc3 ) and nuclear RNase ( Exosc10 ) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3′ regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3′ regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. [ABSTRACT FROM AUTHOR]

Summary Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Summary: Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome. [ABSTRACT FROM AUTHOR]

Summary: Heart failure (HF) is driven by the interplay between regulatory transcription factors and dynamic alterations in chromatin structure. Pathologic gene transactivation in HF is associated with recruitment of histone acetyl-transferases and local chromatin hyperacetylation. We therefore assessed the role 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 expression of genes that are central to HF pathogenesis and relevant to the pathobiology of failing human hearts. This study implicates epigenetic readers as essential effectors of transcriptional pause release during HF pathogenesis and identifies BET coactivator proteins as therapeutic targets in the heart. [ABSTRACT FROM AUTHOR]

Summary: While 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. [Copyright &y& Elsevier]

Summary: Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types. [Copyright &y& Elsevier]

Summary: Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed “Myc target genes.” We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell’s gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation. [ABSTRACT FROM AUTHOR]

Summary: A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception. PaperClip: Display Omitted [ABSTRACT FROM AUTHOR]

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 β-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide. [Display omitted] • CAR-IKZF3 degron expression and CART activity can be regulated in vitro • IMiD treatment controls CART-IKZF3 anti-tumor efficacy in vivo • Such strategy aims to mitigate potential toxicities associated with CART therapy • The IKZF3/lenalidomide pair is portable to other membrane proteins Carbonneau et al. describe a strategy to control the expression levels of membrane proteins of interest, including chimeric antigen receptors displayed in T cells (CARTs). The authors demonstrate that this strategy allows controlling CART anti-tumor activity, in vivo , and discuss the unique features enabling a fast path into clinical development. [ABSTRACT FROM AUTHOR]

Summary Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments. [ABSTRACT FROM AUTHOR]

Summary There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF . YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control. [ABSTRACT FROM AUTHOR]

Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy., Competing Interests: Declaration of interests All authors are past or current employees of Novartis Institutes for Biomedical Research. Some of the authors have patents related to this work: WO2019038717, 3-(1-oxoisoindolin-2-YL)Piperidine-2-6-Dione derivatives and uses thereof (R.E.J.B., S.B., A.C., A.F., and M.V.); and WO2020128972, Dosing regimen and pharmaceutical combinations comprising 3-(1-oxoisoindolin-2-YL)Piperidine-2-6-Dione derivatives (S.B., E.d’H., G.D., R.R.F., D.H., and J.K.-A.)., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance., Competing Interests: Declaration of Interests F.V. receives research funding from Novo Ventures. P.G.R. reports research support from Oncopeptides, Celgene/BMS, and Takeda and is an advisory committee member for Karyopharm, Oncopeptides, Celgene/BMS, Takeda, Janssen, Sanofi, Secura Bio, and GSK. E.S.F. is a founder, science advisory board member, and equity holder in Civetta, Jengu (board member), and Neomorph; an equity holder in C4; and a consultant to Astellas, Novartis, Deerfield, and EcoR1. The Fischer lab receives or has received research funding from Novartis, Astellas, Ajax, and Deerfield. N.P.K. is a consultant to Epiphanes, Inc. W.C.H. is a consultant for Thermo Fisher, Solvasta, MPM Capital, Tyra Biosciences, Jubilant Therapeutics, Frontier Medicines, RAPPTA Therapeutics, and Parexel and is a founder of and advisor to KSQ Therapeutics. L.H.B. receives research funding from AstraZeneca and is a consultant for AstraZeneca and Genentech. B.L.E. has received research funding from Celgene and Deerfield. He has received consulting fees from GRAIL, and he serves on the scientific advisory boards for and holds equity in Skyhawk Therapeutics and Exo Therapeutics. J.E.B. is an author on United States patent applications licensed from the Dana-Farber Cancer Institute to C4 Therapeutics (TPD) and Tensha Therapeutics (now Roche; BRD4 inhibition). He is now an executive of and shareholder in Novartis AG. N.S.G. is a scientific founder, member of the scientific advisory board and equity holder in C4 Therapeutics, Syros Pharmaceuticals, Petra Pharmaceuticals, Ravenna, Inception, Allorion, Jengu, and Soltego (board member) and is the inventor on intellectual property licensed to these entities. R.S., G.M.M., S.G., R.d.M.S., and C.S.M. are authors on a patent application on the use of degraders. C.S.M. also discloses consultant/honoraria from Fate Therapeutics, Ionis Pharmaceuticals and FIMECS; employment of a relative with Takeda; and research funding from Janssen/Johnson & Johnson, TEVA, EMD Serono, Abbvie, Arch Oncology, Karyopharm, Sanofi, and Nurix., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired., Competing Interests: Declaration of Interests M.B. and K.P. received research support and were consultants to the Novartis Institutes for BioMedical Research during the execution of this study. K.P. serves on the scientific advisory board of Farcast Biosciences and Acrivon Therapeutics. M.B. receives sponsored research support from Novartis, serves on the SAB of Kronos Bio, and is a consultant to GTx, Inc., Aleta Biotherapeutics, and H3 Biomedicine. R.Z. and J.B. are current employees of C4 Therapeutics Inc. and Novartis, respectively. C.K. is a scientific founder, fiduciary board of directors member, scientific advisory board member, consultant, and shareholder of Foghorn Therapeutics, Inc. (Cambridge, MA). S. Shu, K.P., and J.B. are inventors of a patent on BET inhibitor resistance that DFCI licensed to Roche., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy., (Published by Elsevier Inc.)

Enhancer profiling is a powerful approach for discovering cis-regulatory elements that define the core transcriptional regulatory circuits of normal and malignant cells. Gene control through enhancer activity is often dominated by a subset of lineage-specific transcription factors. By integrating measures of chromatin accessibility and enrichment for H3K27 acetylation, we have generated regulatory landscapes of chronic lymphocytic leukemia (CLL) samples and representative cell lines. With super enhancer-based modeling of regulatory circuits and assessments of transcription factor dependencies, we discover that the essential super enhancer factor PAX5 dominates CLL regulatory nodes and is essential for CLL cell survival. Targeting enhancer signaling via BET bromodomain inhibition disrupts super enhancer-dependent gene expression with selective effects on CLL core regulatory circuitry, conferring potent anti-tumor activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 6 -methyladenosine (m 6 A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m 6 A/MYC/CEBPA signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

BET inhibitors (BETi) target bromodomain-containing proteins and are currently being evaluated as anti-cancer agents. We find that maximal therapeutic effects of BETi in a Myc-driven B cell lymphoma model required an intact host immune system. Genome-wide analysis of the BETi-induced transcriptional response identified the immune checkpoint ligand Cd274 (Pd-l1) as a Myc-independent, BETi target-gene. BETi directly repressed constitutively expressed and interferon-gamma (IFN-γ) induced CD274 expression across different human and mouse tumor cell lines and primary patient samples. Mechanistically, BETi decreased Brd4 occupancy at the Cd274 locus without any change in Myc occupancy, resulting in transcriptional pausing and rapid loss of Cd274 mRNA production. Finally, targeted inhibition of the PD-1/PD-L1 axis by combining anti-PD-1 antibodies and the BETi JQ1 caused synergistic responses in mice bearing Myc-driven lymphomas. Our data uncover an interaction between BETi and the PD-1/PD-L1 immune-checkpoint and provide mechanistic insight into the transcriptional regulation of CD274., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Restoration of anti-tumor immunity by blocking PD-L1 signaling through the use of antibodies has proven to be beneficial in cancer therapy. Here, we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T-cell-dependent manner. Together, these data demonstrate a small-molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven to be safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3' to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was upregulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET-inhibitor-induced cell death., Competing Interests: The authors have declared that no conflict of interest exists., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

BRD4 governs pathological cardiac gene expression by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for the regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3' UTR of the Brd4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Pol II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Post-translational histone H3 modifications regulate transcriptional competence. The mechanisms by which the epigenome is regulated in response to oncogenic signaling remain unclear. Here we show that H3K4me3 is increased in breast tumors driven by an activated PIK3CA allele and that inhibition of PI3K/AKT signaling reduces promoter-associated H3K4me3 in human breast cancer cells. We show that the H3K4 demethylase KDM5A is an AKT target and that phosphorylation of KDM5A regulates its nuclear localization and promoter occupancy. Supporting a role for KDM5A in mediating PI3K/AKT transcriptional effects, the decreased expression in response to AKT inhibition of a subset of cell-cycle genes associated with poor clinical outcome is blunted by KDM5A silencing. Our data identify a mechanism by which PI3K/AKT signaling modulates the cancer epigenome through controlling H3K4 methylation and suggest that KDM5A subcellular localization and genome occupancy may be pharmacodynamic markers of the activity of PI3K/AKT inhibitors currently in clinical development., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Unrestrained receptor tyrosine kinase (RTK) signaling and epigenetic deregulation are root causes of tumorigenesis. We establish linkage between these processes by demonstrating that aberrant RTK signaling unleashed by oncogenic HRas(G12V) or loss of negative feedback through Sprouty gene deletion remodels histone modifications associated with active typical and super-enhancers. However, although both lesions disrupt the Ras-Erk axis, the expression programs, enhancer signatures, and transcription factor networks modulated upon HRas(G12V) transformation or Sprouty deletion are largely distinct. Oncogenic HRas(G12V) elevates histone 3 lysine 27 acetylation (H3K27ac) levels at enhancers near the transcription factor Gata4 and the kinase Prkcb, as well as their expression levels. We show that Gata4 is necessary for the aberrant gene expression and H3K27ac marking at enhancers, and Prkcb is required for the oncogenic effects of HRas(G12V)-driven cells. Taken together, our findings demonstrate that dynamic reprogramming of the cellular enhancer landscape is a major effect of oncogenic RTK signaling., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation-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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Homeotic (HOX) genes are dysregulated in multiple malignancies, including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to monomethylation (H3K79me1) at HOX loci as hematopoietic cells mature, thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1-to-H3K79me2 conversion is regulated by the DOT1L cofactor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression, and impairs the transforming ability of MLL-AF9, MLL-AF6, and NUP98-NSD1 fusions-mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1-transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Epigenomic 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.

Recurring deletions of chromosome 7 and 7q [-7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ∼50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in -7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human -7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of bromodomain and extra-terminal domain (BET) proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of bromodomain 4 (BRD4) at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL., (Copyright © 2012 Elsevier Inc. All rights reserved.)

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc., (Copyright © 2011 Elsevier Inc. All rights reserved.)