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Steven J Husson - One of the best experts on this subject based on the ideXlab platform.
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the bet family of proteins targets moloney murine leukemia virus integration near transcription start sites
Cell Reports, 2013Co-Authors: Jan De Rijck, Frederic D Bushman, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Bart Landuyt, Steven J HussonAbstract:Summary A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV)-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3, and BRD4) and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins' chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors.
Gerald V Denis - One of the best experts on this subject based on the ideXlab platform.
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BET Protein Function Is Required for Inflammation: BRD2 Genetic Disruption and BET Inhibitor JQ1 Impair Mouse Macrophage Inflammatory Responses
2020Co-Authors: Anna C Belkina, Barbara S Nikolajczyk, Gerald V DenisAbstract:Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are "readers" of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of BRD2 lo mice, a BET protein hypomorph, to show that BRD2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that BRD2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the "cytokine storm" in endotoxemic mice by reducing levels of IL-6 and TNF-a while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production. The Journal of Immunology, 2013, 190: 000-000. R egulation of inflammatory gene expression is tightly controlled through chromatin "readers" that specifically bind histone posttranslational modifications and provide a scaffold, which, in addition to sequence-specific transcription factors, is an integral component of the transcriptional activation complex (1). The biological effects of chromatin-dependent, multiprotein complexes include both transcriptional coactivation and corepression of inflammatory genes in differentiated adult cells (2); thus chromatin readers play critical roles in exquisitely tuned inflammatory responses to a variety of immune system stimuli. Proinflammatory stimuli such as bacterial endotoxin (LPS) arouse extensive transcriptional reprogramming through their ability to activate acetylation of ε-amino groups of nucleosomal histone lysines, a general mark of gene activation (3-5). The acetylated lysine residues are recognized by chromatin readers, many of which contain a conserved structure designated the bromodomain. Bromodomains are highly conserved, left-twisted bundles of four a-helices, with a hydrophobic cleft between two conserved loops that connect the helices (6). The motif uses hydrogen bonding, often at asparagine residues, to bind to acetylated histones (7). In humans, there are at least 40 bromodomain proteins (8, 9), which include histone acetyltransferases, helicases, scaffolding proteins, and other cofactors that control gene transcription. These findings raise the possibility that bromodomain proteins regulate acetylated, histone-packaged inflammatory genes through multiple downstream mechanisms to significantly contribute to outcomes from proinflammatory stimuli. The bromodomain and extraterminal domain (BET) family is a distinct group of bromodomain proteins that in mammals includes BRD2, Brd3, and Brd4, all of which are ubiquitously expressed in mammalian tissues (10-13). BRD2 and Brd4 have been extensively studied in the context of cell-cycle control (14-18) and transcription elongation (19-21), but potential roles in inflammatory responses have not been explored well. Establishing links between BET proteins and inflammation has become clinically critical owing in part to recent drug development efforts, which have shown that drugs able to interrupt interactions between Brd4 and thienodiazepines (22) have efficacy in BET protein-related cancers (23, 24). JQ1 was the first drug developed that specifically interacts with the hydrophobic pocket of the BET bromodomain to block interaction between multiple BET proteins (BRD2/3/4) and acetylated histones (9). JQ1 effectively prompted squamous differentiation and reduced tumor volume of Brd4-dependent, human NUT midline carcinoma xenografts in mice (9), and was proved efficient to block growth of various leukemic cell
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brd4 regulates breast cancer dissemination through jagged1 notch1 signaling
Cancer Research, 2016Co-Authors: Guillaume Andrieu, Anna H Tran, Katherine J Strissel, Gerald V DenisAbstract:The bromodomain and extraterminal (BET) proteins are epigenetic "readers" of acetylated histones in chromatin and have been identified as promising therapeutic targets in diverse cancers. However, it remains unclear how individual family members participate in cancer progression and small molecule inhibitors such as JQ1 can target functionally independent BET proteins. Here, we report a signaling pathway involving BRD4 and the ligand/receptor pair Jagged1/Notch1 that sustains triple-negative breast cancer migration and invasion. BRD4, but not BRD2 or BRD3, regulated Jagged1 expression and Notch1 signaling. BRD4-selective knockdown suppressed Notch1 activity and impeded breast cancer migration and invasion. BRD4 was required for IL6-stimulated, Notch1-induced migration and invasion, coupling microenvironment inflammation with cancer propagation. Moreover, in patients, BRD4 and Jagged1 expression positively correlated with the presence of distant metastases. These results identify a BRD4/Jagged1/Notch1 signaling pathway that is critical for dissemination of triple-negative breast cancer. Cancer Res; 76(22); 6555-67. ©2016 AACR.
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clinical trials for bet inhibitors run ahead of the science
Drug Discovery Today: Technologies, 2016Co-Authors: Guillaume Andrieu, Anna C Belkina, Gerald V DenisAbstract:Several cancer clinical trials for small molecule inhibitors of BET bromodomain proteins have been initiated. There is enthusiasm for the anti-proliferative effect of inhibiting BRD4, one of the targets of these inhibitors, which is thought to cooperate with MYC, a long-desired target for cancer therapeutics. However, no current inhibitor is selective for BRD4 among the three somatic BET proteins, which include BRD2 and BRD3; their respective functions are partially overlapping and none are functionally redundant with BRD4. Each BET protein controls distinct transcriptional pathways that are important for functions beyond cancer cell proliferation, including insulin production, cytokine gene transcription, T cell differentiation, adipogenesis and most seriously, active repression of dangerous latent viruses like HIV. BET inhibitors have been shown to reactivate HIV in human cells. Failure to appreciate that at concentrations used, no available BET inhibitor is member-selective, or to develop a sound biological basis to understand the diverse functions of BET proteins before undertaking for these clinical trials is reckless and likely to lead to adverse events. More mechanistic information from new basic science studies should enable proper focus on the most relevant cancers and define the expected side effect profiles.
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bet protein function is required for inflammation BRD2 genetic disruption and bet inhibitor jq1 impair mouse macrophage inflammatory responses
Journal of Immunology, 2013Co-Authors: Anna C Belkina, Barbara S Nikolajczyk, Gerald V DenisAbstract:Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are "readers" of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of BRD2 lo mice, a BET protein hypomorph, to show that BRD2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that BRD2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the "cytokine storm" in endotoxemic mice by reducing levels of IL-6 and TNF-α while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production.
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kaposi s sarcoma associated herpesvirus latency associated nuclear antigen interacts with bromodomain protein brd4 on host mitotic chromosomes
Journal of Virology, 2006Co-Authors: Jianxin You, Gerald V Denis, Viswanathan Srinivasan, William J Harrington, Mary E Ballestas, Kenneth M KayeAbstract:The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/BRD2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/BRD2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.
Alessio Ciulli - One of the best experts on this subject based on the ideXlab platform.
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SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate.
ACS Chemical Biology, 2019Co-Authors: Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio CiulliAbstract:Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins BRD2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs t...
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spr measured dissociation kinetics of protac ternary complexes influence target degradation rate
bioRxiv, 2019Co-Authors: Michael J Roy, Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio CiulliAbstract:ABSTRACT Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins BRD2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or BRD2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of BRD2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.
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SPR-measured kinetics of PROTAC ternary complexes influence target degradation rate
bioRxiv, 2018Co-Authors: Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio CiulliAbstract:Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, via a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no method to monitor ternary complex kinetics has been reported, and it remains to be addressed how this might impact on the kinetics of protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins BRD2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that form either cooperative or non-cooperative complexes. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or BRD2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of BRD2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.
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selective small molecule induced degradation of the bet bromodomain protein brd4
ACS Chemical Biology, 2015Co-Authors: Michael Zengerle, Kwokho Chan, Alessio CiulliAbstract:The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent wit...
Hidayatullah G Munshi - One of the best experts on this subject based on the ideXlab platform.
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bet inhibitors block pancreatic stellate cell collagen i production and attenuate fibrosis in vivo
JCI insight, 2017Co-Authors: Krishan Kumar, Brian Decant, Paul J Grippo, David J Bentrem, Kazumi Ebine, Rosa F. Hwang, Hidayatullah G MunshiAbstract:The fibrotic reaction, which can account for over 70%-80% of the tumor mass, is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen I production and fibrosis in vivo. In this report, we show that members of the bromodomain and extraterminal (BET) family of proteins are expressed in primary PSCs isolated from human PDAC tumors, with BRD4 positively regulating, and BRD2 and BRD3 negatively regulating, collagen I expression in primary cancer-associated PSCs. We show that the inhibitory effect of pan-BET inhibitors on collagen I expression in primary cancer-associated PSCs is through blocking of BRD4 function. Importantly, we show that FOSL1 is repressed by BRD4 in primary cancer-associated PSCs and negatively regulates collagen I expression. While BET inhibitors do not affect viability or induce PSC apoptosis or senescence, BET inhibitors induce primary cancer-associated PSCs to become quiescent. Finally, we show that BET inhibitors attenuate stellate cell activation, fibrosis, and collagen I production in the EL-KrasG12D transgenic mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis by modulating the activation and function of cancer-associated PSCs.
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abstract b17 bet inhibitors block pancreatic stellate cell collagen production and attenuate fibrosis in vivo
Cancer Research, 2016Co-Authors: Krishan Kumar, Brian Decant, Hattaway Z Hattaway, Paul J Grippo, David J Bentrem, Kazumi Ebine, Hidayatullah G MunshiAbstract:The fibrotic reaction, which can account for over 70% of the tumor mass, is a characteristic feature of the majority of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen production and fibrosis in vivo. In this report we show that bromodomain-containing protein 4 (BRD4), a member of bromodomain and extraterminal (BET) family of proteins, is expressed in activated PSCs in human PDAC tumors. We also show that BET inhibitors and specific targeting of BRD4, in contrast to targeting of BRD2 and BRD3, decrease collagen production by primary PSCs isolated from human PDAC tumors. While BET inhibitors do not induce apoptosis of primary PSCs or affect α-SMA expression, BET inhibitors disrupt α-SMA filament organization in stellate cells. Additionally, BET inhibitors attenuate stellate cell activation, fibrosis and collagen production in vivo in a Kras mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis partly by modulating PSC activation and function. Citation Format: Krishan Kumar, Brian DeCant, Hattaway Z. Hattaway, Paul J. Grippo, David J. Bentrem, Kazumi Ebine, Hidayatullah G. Munshi.{Authors}. BET inhibitors block pancreatic stellate cell collagen production and attenuate fibrosis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B17.
Zeger Debyser - One of the best experts on this subject based on the ideXlab platform.
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bromodomain and extra terminal bet proteins target moloney murine leukemia virus integration to transcription start sites
Retrovirology, 2013Co-Authors: Jan De Riick, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Katrien Busschots, Steven Husson, Rik Gijsbers, Zeger DebyserAbstract:A hallmark of retroviral replication is stable integration of the viral genome in the host cell DNA. This characteristic makes retroviral-derived vector particles attractive vehicles for gene therapy. However, retroviral integration is not a random process. Lentiviruses preferentially integrate in the body of active transcription units, while gammaretroviruses, including Moloney Murine Leukemia Virus (MLV), favour transcription start sites and CpG islands. In clinical trials using gammaretroviral vectors for gene therapy, leukemogenesis has been associated with integration of vectors near oncogene transcription start sites. We found that the bromodomain and extra-terminal (BET) proteins (BRD2, BRD3 and BRD4) interact with MLV integrase and direct integration towards transcription start regions. BET proteins specifically bind and co-localize with the gammaretrovirus integrase protein in the nucleus of the cell. The interaction is gammaretroviral-specific and mediated by the integrase C-terminal domain and the BET extraterminal (ET) domain as determined by co-immunoprecipitation assays and in an Alphascreen assay using recombinant proteins. Interfering with chromatin interaction of BET proteins via specific bromodomain inhibitors JQ1 and l-BET decreases MLV virus replication and MLV vector transduction 5-to 10-fold, while HIV vector transduction is not affected. Analysis of viral DNA intermediates by quantitative PCR revealed a block at the integration step. In addition, bromodomain inhibitors do not have an effect on the late steps of viral replication. MLV integration site distribution analysis revealed a strong correlation with the BET protein chromatin binding profile. Finally, expression of an artificial fusion protein that merges the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration into the body of actively transcribed genes, paralleling the Human Immunodeficiency Virus (HIV) integration pattern. Our results explain the molecular mechanism behind gammaretroviral integration site targeting and suggest methods for engineering gammaretroviral vectors with a safer integration site profile.