Publications by authors named "James E Bradner"

233 Publications

Selective targeting of MYC mRNA by stabilized antisense oligonucleotides.

Oncogene 2021 Oct 14. Epub 2021 Oct 14.

Broad Institute of MIT & Harvard, Cambridge, MA, 02142, USA.

MYC is a prolific proto-oncogene driving the malignant behaviors of numerous common cancers, yet potent and selective cell-permeable inhibitors of MYC remain elusive. In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC translation. Here we describe a series of conformationally stabilized synthetic antisense oligonucleotides designed to target MYC mRNA (MYCASOs). To support bioactivity, we designed and synthesized this focused library of MYCASOs incorporating locked nucleic acid (LNA) bases at the 5'- and 3'-ends, a phosphorothioate backbone, and internal DNA bases. Treatment of MYC-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 5' Rapid Amplification of cDNA Ends (RACE) assay. MYCASO treatment of cancer cell lines leads to significant inhibition of cellular proliferation while specifically perturbing MYC-driven gene expression signatures. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment decreases MYC protein levels within tumors, decreases tumor burden, and improves overall survival. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of MYC activity, and promising therapeutic agents for MYC-addicted tumors.
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http://dx.doi.org/10.1038/s41388-021-02053-4DOI Listing
October 2021

Correction: HDAC6 inhibitor WT161 downregulates growth factor receptors in breast cancer.

Oncotarget 2021 Aug 17;12(17):1736. Epub 2021 Aug 17.

Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.

[This corrects the article DOI: 10.18632/oncotarget.19019.].
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http://dx.doi.org/10.18632/oncotarget.28051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378766PMC
August 2021

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

J Clin Invest 2021 Aug;131(16)

Center for Cellular Immunotherapies.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.
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http://dx.doi.org/10.1172/JCI145459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363276PMC
August 2021

The synergy of BET inhibitors with aurora A kinase inhibitors in MYCN-amplified neuroblastoma is heightened with functional TP53.

Neoplasia 2021 06 7;23(6):624-633. Epub 2021 Jun 7.

Department of Pediatrics, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA. Electronic address:

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.
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http://dx.doi.org/10.1016/j.neo.2021.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192452PMC
June 2021

Targeting BRD4 in acute myeloid leukemia with partial tandem duplication of the gene.

Haematologica 2021 Sep 1;106(9):2527-2532. Epub 2021 Sep 1.

The Ohio State University, Comprehensive Cancer Center, Columbus, OH, USA; Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH.

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http://dx.doi.org/10.3324/haematol.2020.271627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409020PMC
September 2021

Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer.

Sci Adv 2021 Jan 15;7(3). Epub 2021 Jan 15.

Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a "hit" compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.
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http://dx.doi.org/10.1126/sciadv.abc4897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810372PMC
January 2021

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Cell Rep 2021 01;34(1):108532

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.

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.
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http://dx.doi.org/10.1016/j.celrep.2020.108532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485877PMC
January 2021

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

Cell Chem Biol 2021 06 16;28(6):802-812.e6. Epub 2020 Dec 16.

Novartis Institutes for Biomedical Research, Cambridge, MA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chembiol.2020.11.012DOI Listing
June 2021

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

J Clin Invest 2020 11;130(11):5875-5892

Division of Cancer Therapeutics, Institute of Cancer Research (ICR), London and Royal Marsden NHS Trust, Sutton, United Kingdom.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.
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http://dx.doi.org/10.1172/JCI134132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598076PMC
November 2020

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules.

Nat Commun 2020 09 18;11(1):4687. Epub 2020 Sep 18.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAG-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12-tagged proteins. dTAG-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.
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http://dx.doi.org/10.1038/s41467-020-18377-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501296PMC
September 2020

BET bromodomain proteins regulate transcriptional reprogramming in genetic dilated cardiomyopathy.

JCI Insight 2020 08 6;5(15). Epub 2020 Aug 6.

Emory University School of Medicine, Atlanta, Georgia, USA.

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) - a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLNR9C or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB-mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.
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http://dx.doi.org/10.1172/jci.insight.138687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455078PMC
August 2020

Selective Mediator dependence of cell-type-specifying transcription.

Nat Genet 2020 07 1;52(7):719-727. Epub 2020 Jun 1.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.
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http://dx.doi.org/10.1038/s41588-020-0635-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610447PMC
July 2020

Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation.

Nat Commun 2020 05 7;11(1):2243. Epub 2020 May 7.

Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany.

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.
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http://dx.doi.org/10.1038/s41467-020-16103-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206058PMC
May 2020

Dual Inhibition of TAF1 and BET Bromodomains from the BI-2536 Kinase Inhibitor Scaffold.

ACS Med Chem Lett 2019 Oct 13;10(10):1443-1449. Epub 2019 Sep 13.

Department of Cancer Biology, Dana Farber Cancer Institute, Boston Massachusetts 02115, United States.

Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792166PMC
October 2019

High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program.

Nat Commun 2019 09 25;10(1):4358. Epub 2019 Sep 25.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Systemic metabolic alterations associated with increased consumption of saturated fat and obesity are linked with increased risk of prostate cancer progression and mortality, but the molecular underpinnings of this association are poorly understood. Here, we demonstrate in a murine prostate cancer model, that high-fat diet (HFD) enhances the MYC transcriptional program through metabolic alterations that favour histone H4K20 hypomethylation at the promoter regions of MYC regulated genes, leading to increased cellular proliferation and tumour burden. Saturated fat intake (SFI) is also associated with an enhanced MYC transcriptional signature in prostate cancer patients. The SFI-induced MYC signature independently predicts prostate cancer progression and death. Finally, switching from a high-fat to a low-fat diet, attenuates the MYC transcriptional program in mice. Our findings suggest that in primary prostate cancer, dietary SFI contributes to tumour progression by mimicking MYC over expression, setting the stage for therapeutic approaches involving changes to the diet.
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http://dx.doi.org/10.1038/s41467-019-12298-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761092PMC
September 2019

PAX8 activates metabolic genes via enhancer elements in Renal Cell Carcinoma.

Nat Commun 2019 08 20;10(1):3739. Epub 2019 Aug 20.

Disease Area Oncology, Novartis Institute for Biomedical Research, Basel, Switzerland.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.
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http://dx.doi.org/10.1038/s41467-019-11672-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702156PMC
August 2019

Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma.

Nat Commun 2019 06 3;10(1):2400. Epub 2019 Jun 3.

Department of Pathology, Brigham and Women's Hospital, Boston, USA.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.
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http://dx.doi.org/10.1038/s41467-019-10307-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546744PMC
June 2019

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

Nat Genet 2019 06 27;51(6):990-998. Epub 2019 May 27.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
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http://dx.doi.org/10.1038/s41588-019-0413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952269PMC
June 2019

The BET inhibitor JQ1 attenuates double-strand break repair and sensitizes models of pancreatic ductal adenocarcinoma to PARP inhibitors.

EBioMedicine 2019 Jun 22;44:419-430. Epub 2019 May 22.

Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1670 University Blvd, Birmingham, AL, USA. Electronic address:

Background: DNA repair deficiency accumulates DNA damage and sensitizes tumor cells to PARP inhibitors (PARPi). Based on our observation that the BET inhibitor JQ1 increases levels of DNA damage, we evaluated the efficacy of JQ1 + the PARPi olaparib in preclinical models of pancreatic ductal adenocarcinoma (PDAC). We also addressed the mechanism by which JQ1 increased DNA damage.

Methods: The effect of JQ1 + olaparib on in vivo tumor growth was assessed with patient-derived xenograft (PDX) models of PDAC. Changes in protein expression were detected by immunohistochemistry and immunoblot. In vitro growth inhibition and mechanistic studies were done using alamarBlue, qRT-PCR, immunoblot, immunofluorescence, ChIP, and shRNA knockdown assays.

Findings: Tumors exposed in vivo to JQ1 had higher levels of the DNA damage marker γH2AX than tumors exposed to vehicle only. Increases in γH2AX was concomitant with decreased expression of DNA repair proteins Ku80 and RAD51. JQ1 + olaparib inhibited the growth of PDX tumors greater than either drug alone. Mechanistically, ChIP assays demonstrated that JQ1 decreased the association of BRD4 and BRD2 with promoter loci of Ku80 and RAD51, and shRNA data showed that expression of Ku80 and RAD51 was BRD4- and BRD2-dependent in PDAC cell lines.

Interpretation: The data are consistent with the hypothesis that JQ1 confers a repair deficient phenotype and the consequent accumulation of DNA damage sensitizes PDAC cells to PARPi. Combinations of BET inhibitors with PARPi may provide a novel strategy for treating PDAC. FUND: NIH grants R01CA208272 and R21CA205501; UAB CMB T32 predoctoral training grant.
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http://dx.doi.org/10.1016/j.ebiom.2019.05.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604668PMC
June 2019

Small-molecule targeting of brachyury transcription factor addiction in chordoma.

Nat Med 2019 02 21;25(2):292-300. Epub 2019 Jan 21.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Chordoma is a primary bone cancer with no approved therapy. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.
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http://dx.doi.org/10.1038/s41591-018-0312-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633917PMC
February 2019

Enhancer Domains in Gastrointestinal Stromal Tumor Regulate KIT Expression and Are Targetable by BET Bromodomain Inhibition.

Cancer Res 2019 03 10;79(5):994-1009. Epub 2019 Jan 10.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the enhancer essential for gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the enhancer domain and reduced gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects and , with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST. SIGNIFICANCE: Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397693PMC
March 2019

Non-overlapping Control of Transcriptome by Promoter- and Super-Enhancer-Associated Dependencies in Multiple Myeloma.

Cell Rep 2018 12;25(13):3693-3705.e6

Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA; VA Boston Healthcare System, Boston, MA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407615PMC
December 2018

Enhancer Architecture and Essential Core Regulatory Circuitry of Chronic Lymphocytic Leukemia.

Cancer Cell 2018 12 29;34(6):982-995.e7. Epub 2018 Nov 29.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT & Harvard, Cambridge, MA 02142, USA; Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ccell.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298230PMC
December 2018

Targeted degradation of BRD9 reverses oncogenic gene expression in synovial sarcoma.

Elife 2018 11 15;7. Epub 2018 Nov 15.

Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston Children's Hospital and Harvard Medical School, Boston, United States.

Synovial sarcoma tumours contain a characteristic fusion protein, SS18-SSX, which drives disease development. Targeting oncogenic fusion proteins presents an attractive therapeutic opportunity. However, SS18-SSX has proven intractable for therapeutic intervention. Using a domain-focused CRISPR screen we identified the bromodomain of BRD9 as a critical functional dependency in synovial sarcoma. BRD9 is a component of SS18-SSX containing BAF complexes in synovial sarcoma cells; and integration of BRD9 into these complexes is critical for cell growth. Moreover BRD9 and SS18-SSX co-localize extensively on the synovial sarcoma genome. Remarkably, synovial sarcoma cells are highly sensitive to a novel small molecule degrader of BRD9, while other sarcoma subtypes are unaffected. Degradation of BRD9 induces downregulation of oncogenic transcriptional programs and inhibits tumour progression in vivo. We demonstrate that BRD9 supports oncogenic mechanisms underlying the SS18-SSX fusion in synovial sarcoma and highlight targeted degradation of BRD9 as a potential therapeutic opportunity in this disease.
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http://dx.doi.org/10.7554/eLife.41305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277197PMC
November 2018

A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation.

Nat Cell Biol 2018 12 5;20(12):1410-1420. Epub 2018 Nov 5.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.

Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.
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http://dx.doi.org/10.1038/s41556-018-0221-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698386PMC
December 2018

Small-molecule BCL6 inhibitor effectively treats mice with nonsclerodermatous chronic graft-versus-host disease.

Blood 2019 01 2;133(1):94-99. Epub 2018 Oct 2.

Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN.

Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.
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http://dx.doi.org/10.1182/blood-2018-03-839993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318432PMC
January 2019

Structure-Guided Design and Development of Potent and Selective Dual Bromodomain 4 (BRD4)/Polo-like Kinase 1 (PLK1) Inhibitors.

J Med Chem 2018 09 30;61(17):7785-7795. Epub 2018 Aug 30.

Department of Chemistry , University of Massachusetts-Boston , Boston , Massachusetts 02125 , United States.

The simultaneous inhibition of polo-like kinase 1 (PLK1) and BRD4 bromodomain by a single molecule could lead to the development of an effective therapeutic strategy for a variety of diseases in which PLK1 and BRD4 are implicated. Compound 23 has been found to be a potent dual kinase-bromodomain inhibitor (BRD4-BD1 IC = 28 nM, PLK1 IC = 40 nM). Compound 6 was found to be the most selective PLK1 inhibitor over BRD4 in our series (BRD4-BD1 IC = 2579 nM, PLK1 IC = 9.9 nM). Molecular docking studies with 23 and BRD4-BD1/PLK1 as well as with 6 corroborate the biochemical assay results.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309379PMC
September 2018

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains.

ACS Chem Biol 2018 09 31;13(9):2438-2448. Epub 2018 Aug 31.

Department of Cancer Biology , Dana-Farber Cancer Institute , Boston , Massachusetts 02215 , United States.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.
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http://dx.doi.org/10.1021/acschembio.7b00638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333101PMC
September 2018
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