RESUMO
BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.
Assuntos
Apoptose , Neoplasias Ósseas , Proteínas de Ciclo Celular , Proliferação de Células , Osteossarcoma , Fatores de Transcrição , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Nus , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The antitumor performance of PROteolysis-TArgeting Chimeras (PROTACs) is limited by its insufficient tumor specificity and poor pharmacokinetics. These disadvantages are further compounded by tumor heterogeneity, especially the presence of cancer stem-like cells, which drive tumor growth and relapse. Herein, we design a region-confined PROTAC nanoplatform that integrates both reactive oxygen species (ROS)-activatable and hypoxia-responsive PROTAC prodrugs for the precise manipulation of bromodomain and extraterminal protein 4 expression and tumor eradication. These PROTAC nanoparticles selectively accumulate within and penetrate deep into tumors via response to matrix metalloproteinase-2. Photoactivity is then reactivated in response to the acidic intracellular milieu and the PROTAC is discharged due to the ROS generated via photodynamic therapy specifically within the normoxic microenvironment. Moreover, the latent hypoxia-responsive PROTAC prodrug is restored in hypoxic cancer stem-like cells overexpressing nitroreductase. Here, we show the ability of region-confined PROTAC nanoplatform to effectively degrade BRD4 in both normoxic and hypoxic environments, markedly hindering tumor progression in breast and head-neck tumor models.
Assuntos
Proteínas de Ciclo Celular , Nanopartículas , Proteólise , Fatores de Transcrição , Humanos , Proteólise/efeitos dos fármacos , Animais , Nanopartículas/química , Linhagem Celular Tumoral , Camundongos , Fatores de Transcrição/metabolismo , Feminino , Proteínas de Ciclo Celular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Fotoquimioterapia/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Microambiente Tumoral/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Nucleares/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Proteínas que Contêm BromodomínioRESUMO
Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Redes Reguladoras de Genes , Fatores de Transcrição , Transcrição Gênica , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Complexo Mediador/genética , Análise de Célula Única , Transcriptoma , Coesinas , Proteínas que Contêm BromodomínioRESUMO
The antiapoptotic protein BCL2A1 is highly, but very heterogeneously expressed in Diffuse Large B-cell Lymphoma (DLBCL). Particularly in the context of resistance to current therapies, BCL2A1 appears to play an important role in protecting cancer cells from the induction of cell death. Reducing BCL2A1 levels may have therapeutic potential, however, no specific inhibitor is currently available. In this study, we hypothesized that the signaling network regulated by epigenetic readers may regulate the transcription of BCL2A1 and hence that inhibition of Bromodomain and Extra-Terminal (BET) proteins may reduce BCL2A1 expression thus leading to cell death in DLBCL cell lines. We found that the mechanisms of action of acetyl-lysine competitive BET inhibitors are different from those of proteolysis targeting chimeras (PROTACs) that induce the degradation of BET proteins. Both classes of BETi reduced the expression of BCL2A1 which coincided with a marked downregulation of c-MYC. Mechanistically, BET inhibition attenuated the constitutively active canonical nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway and inhibited p65 activation. Furthermore, signal transducer of activated transcription (STAT) signaling was reduced by inhibiting BET proteins, targeting another pathway that is often constitutively active in DLBCL. Both pathways were also inhibited by the IκB kinase inhibitor TPCA-1, resulting in decreased BCL2A1 and c-MYC expression. Taken together, our study highlights a novel complex regulatory network that links BET proteins to both NFκB and STAT survival signaling pathways controlling both BCL2A1 and c-MYC expression in DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas que Contêm Bromodomínio , Proteínas , Antígenos de Histocompatibilidade MenorRESUMO
Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.
Assuntos
Microscopia Crioeletrônica , Lisina Acetiltransferase 5 , Humanos , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/química , Lisina Acetiltransferase 5/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , DNA Helicases/metabolismo , DNA Helicases/química , Modelos Moleculares , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Ligação Proteica , Multimerização Proteica , Proteínas que Contêm Bromodomínio , Proteínas Adaptadoras de Transdução de SinalRESUMO
We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular , Bibliotecas de Moléculas Pequenas , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proliferação de Células/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Azepinas/farmacologia , Azepinas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Indolizinas/química , Indolizinas/farmacologia , Linhagem Celular Tumoral , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ligantes , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/síntese química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas que Contêm Bromodomínio , Óxidos N-Cíclicos , Compostos de PiridínioRESUMO
The bromodomain-containing protein BRD9 has emerged as an attractive therapeutic target. In the present study, we successfully identified a number of highly potent BRD9 degraders by using two different cereblon ligands developed in our laboratory. Further optimization led to the discovery of CW-3308 as a potent, selective, and orally bioavailable BRD9 degrader. It displayed degradation potency (DC50) < 10 nM and efficiency (Dmax) > 90% against BRD9 in the G401 rhabdoid tumor and HS-SY-II synovial sarcoma cell lines and had a high degradation selectivity over BRD7 and BRD4 proteins. CW-3308 achieved 91% of oral bioavailability in mice. A single oral dose efficiently reduced the BRD9 protein by >90% in the synovial sarcoma HS-SY-II xenograft tumor tissue. Oral administration effectively inhibited HS-SY-II xenograft tumor growth in mice. CW-3308 is a promising lead compound for further optimization and extensive evaluation for the treatment of synovial sarcoma, rhabdoid tumor, and other BRD9-dependent human diseases.
Assuntos
Fatores de Transcrição , Humanos , Animais , Administração Oral , Camundongos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Proteólise/efeitos dos fármacos , Descoberta de Drogas , Camundongos Nus , Disponibilidade Biológica , Proteínas que Contêm BromodomínioRESUMO
Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Fatores de Transcrição , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linhagem da Célula/genética , Células Cultivadas , Análise de Célula Única , Proteínas que Contêm BromodomínioRESUMO
In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.
Assuntos
Proteínas de Ciclo Celular , Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Fatores de Transcrição , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Separação de Fases , Proteínas que Contêm BromodomínioRESUMO
Bromodomain-containing protein 9 (BRD9) is a key player in chromatin remodeling and gene expression regulation, and it is closely associated with the development of various diseases, including cancers. Recent studies have indicated that inhibition of BRD9 may have potential value in the treatment of certain cancers. Molecular dynamics (MD) simulations, Markov modeling and principal component analysis were performed to investigate the binding mechanisms of allosteric inhibitor POJ and orthosteric inhibitor 82I to BRD9 and its allosteric regulation. Our results indicate that binding of these two types of inhibitors induces significant structural changes in the protein, particularly in the formation and dissolution of α-helical regions. Markov flux analysis reveals notable changes occurring in the α-helicity near the ZA loop during the inhibitor binding process. Calculations of binding free energies reveal that the cooperation of orthosteric and allosteric inhibitors affects binding ability of inhibitors to BRD9 and modifies the active sites of orthosteric and allosteric positions. This research is expected to provide new insights into the inhibitory mechanism of 82I and POJ on BRD9 and offers a theoretical foundation for development of cancer treatment strategies targeting BRD9.
Assuntos
Cadeias de Markov , Simulação de Dinâmica Molecular , Ligação Proteica , Fatores de Transcrição , Regulação Alostérica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/antagonistas & inibidores , Humanos , Sítios de Ligação , Análise de Componente Principal , Termodinâmica , Proteínas que Contêm BromodomínioRESUMO
Under subarachnoid hemorrhage (SAH) conditions, astrocytes undergo a marked intensification of glycolytic activity, resulting in the generation of substantial amounts of lactate to maintain the energy demand for neurons and other brain cells. Lactate has garnered increasing attention in recent years because of its emerging role in critical biological processes such as inflammation regulation and neuroprotection, particularly through its histone lactylation. Bromodomain-containing protein 4 (BRD4) plays a crucial role in maintaining neural development and promoting memory formation in the central nervous system. Nonetheless, the function and regulatory mechanism of BRD4 and histone lactylation in astrocytes following SAH remain elusive. Our findings indicate that BRD4, a crucial epigenetic regulator, plays a definitive role in histone lactylation. Both in vitro and in vivo, these results demonstrated that targeted silencing of BRD4 in astrocytes can significantly reduce H4K8la lactylation, thereby aggravating the A1 polarization of astrocytes and ultimately affecting the recovery of neural function and prognosis in mice after SAH. In summary, BRD4 plays a pivotal role in modulating astrocyte polarization following SAH via histone lactylation. Targeting this mechanism might offer an efficient therapeutic strategy for SAH.
Assuntos
Astrócitos , Proteínas que Contêm Bromodomínio , Histonas , Hemorragia Subaracnóidea , Fatores de Transcrição , Animais , Masculino , Camundongos , Astrócitos/metabolismo , Proteínas que Contêm Bromodomínio/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Histonas/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The maintenance of nuclear shape is essential for cellular homeostasis and disruptions in this process have been linked to various pathological conditions, including cancer, laminopathies, and aging. Despite the significance of nuclear shape, the precise molecular mechanisms controlling it are not fully understood. In this study, we have identified the YEATS domain-containing protein 4 (GAS41) as a previously unidentified factor involved in regulating nuclear morphology. Genetic ablation of GAS41 in colorectal cancer cells resulted in significant abnormalities in nuclear shape and inhibited cancer cell proliferation both in vitro and in vivo. Restoration experiments revealed that wild-type GAS41, but not a YEATS domain mutant devoid of histone H3 lysine 27 acetylation or crotonylation (H3K27ac/cr) binding, rescued the aberrant nuclear phenotypes in GAS41-deficient cells, highlighting the importance of GAS41's binding to H3K27ac/cr in nuclear shape regulation. Further experiments showed that GAS41 interacts with H3K27ac/cr to regulate the expression of key nuclear shape regulators, including LMNB1, LMNB2, SYNE4, and LEMD2. Mechanistically, GAS41 recruited BRD2 and the Mediator complex to gene loci of these regulators, promoting their transcriptional activation. Disruption of GAS41-H3K27ac/cr binding caused BRD2, MED14 and MED23 to dissociate from gene loci, leading to nuclear shape abnormalities. Overall, our findings demonstrate that GAS41 collaborates with BRD2 and the Mediator complex to control the expression of crucial nuclear shape regulators.
Assuntos
Núcleo Celular , Neoplasias Colorretais , Fatores de Transcrição , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Linhagem Celular Tumoral , Proliferação de Células , Camundongos , Histonas/metabolismo , Camundongos Nus , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Células HCT116 , Proteínas que Contêm BromodomínioRESUMO
Upon infection, naive CD8+ T cells differentiate into cytotoxic effector cells to eliminate the pathogen-infected cells. Although many mechanisms underlying this process have been demonstrated, the regulatory role of chromatin remodeling system in this process remains largely unknown. Here we show that BRD7, a component of the polybromo-associated BAF complex (PBAF), was required for naive CD8+ T cells to differentiate into functional short-lived effector cells (SLECs) in response to acute infections caused by influenza virus or lymphocytic choriomeningitis virus (LCMV). BRD7 deficiency in CD8+ T cells resulted in profound defects in effector population and functions, thereby impairing viral clearance and host recovery. Further mechanical studies indicate that the expression of BRD7 significantly turned to high from naive CD8+ T cells to effector cells, which bridged BRG1 and PBRM1 to the core module of PBAF complex, consequently facilitating the assembly of PBAF complex rather than BAF complex in the effector cells. The PBAF complex changed the chromatin accessibility at the loci of Tbx21 gene and upregulated its expression, leading to the maturation of effector T cells. Our research demonstrates that BRD7 and the PBAF complex are key in CD8+ T cell development and present a significant target for advancing immune therapies.
Assuntos
Linfócitos T CD8-Positivos , Diferenciação Celular , Proteínas Cromossômicas não Histona , Vírus da Coriomeningite Linfocítica , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Diferenciação Celular/imunologia , Diferenciação Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Vírus da Coriomeningite Linfocítica/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Montagem e Desmontagem da Cromatina , Proteínas que Contêm BromodomínioRESUMO
The BET (bromodomain and extraterminal domain) family of proteins, particularly BRD4 (bromodomain-containing protein 4), plays a crucial role in transcription regulation and epigenetic mechanisms, impacting key cellular processes such as proliferation, differentiation, and the DNA damage response. BRD4, the most studied member of this family, binds to acetylated lysines on both histones and non-histone proteins, thereby regulating gene expression and influencing diverse cellular functions such as the cell cycle, tumorigenesis, and immune responses to viral infections. Given BRD4's involvement in these fundamental processes, it is implicated in various diseases, including cancer and inflammation, making it a promising target for therapeutic development. This review comprehensively explores the roles of the BET family in gene transcription, DNA damage response, and viral infection, discussing the potential of targeted small-molecule compounds and highlighting BET proteins as promising candidates for anticancer therapy.
Assuntos
Epigênese Genética , Neoplasias , Fatores de Transcrição , Viroses , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Viroses/metabolismo , Viroses/genética , Viroses/virologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animais , Domínios Proteicos , Dano ao DNA , Histonas/metabolismo , Proteínas que Contêm BromodomínioRESUMO
Histone deacetylase inhibitors (HDACis) show beneficial effects on different hematological malignancy subtypes. However, their impacts on treating solid tumors are still limited due to diverse resistance mechanisms. Recent studies have found that the feedback activation of BRD4-LIFR-JAK1-STAT3 pathway after HDACi incubation is a vital mechanism inducing resistance of specific solid tumor cells to HDACis. This review summarizes the recent development of multi-target HDACis that can concurrently block BRD4-LIFR-JAK1-STAT3 pathway. Moreover, our findings hope to shed novel lights on developing novel multi-target HDACis with reduced BRD4-LIFR-JAK1-STAT3-mediated drug resistance in some tumors.
Assuntos
Inibidores de Histona Desacetilases , Janus Quinase 1 , Neoplasias , Fator de Transcrição STAT3 , Transdução de Sinais , Fatores de Transcrição , Humanos , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/síntese química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas que Contêm BromodomínioRESUMO
Purpose: Ocular melanoma is a common primary malignant ocular tumor in adults with limited effective treatments. Epigenetic regulation plays an important role in tumor development. The switching/sucrose nonfermentation (SWI/SNF) chromatin remodeling complex and bromodomain and extraterminal domain family proteins are epigenetic regulators involved in several cancers. We aimed to screen a candidate small molecule inhibitor targeting these regulators and investigate its effect and mechanism in ocular melanoma. Methods: We observed phenotypes caused by knockdown of the corresponding gene and synergistic effects with BRD inhibitor treatment and SWI/SNF complex knockdown. The effect of JQ-1 on ocular melanoma cell cycle and apoptosis was analyzed with flow cytometry. Via RNA sequencing, we also explored the mechanism of BRD4. Results: The best tumor inhibitory effect was observed for the BRD4 inhibitor (JQ-1), although there were no statistically obvious changes in the shBRD4 and shBRD9 groups. Interestingly, the inhibitory effect of JQ-1 was decrease in the shBRD4 group. JQ-1 inhibits the growth of melanoma in various cell lines and in tumor-bearing mice. We found 17 of these 28 common differentially expressed genes were downregulated after MEL270 and MEL290 cells treated with JQ-1. Four of these 17 genes, TP53I11, SH2D5, SEMA5A, and MDGA1, were positively correlated with BRD4. In TCGA database, low expression of TP53I11, SH2D5, SEMA5A, and MDGA1 improved the overall survival rate of patients. Furthermore, the disease-free survival rate was increased in the groups with low expression of TP53I11, SH2D5, and SEMA5A. Conclusions: JQ-1 may act downstream of BRD4 and suppress ocular melanoma growth by inducing G1 cell cycle arrest.
Assuntos
Apoptose , Azepinas , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Melanoma , Fatores de Transcrição , Triazóis , Animais , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Camundongos , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Azepinas/farmacologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Neoplasias Uveais/metabolismo , Citometria de Fluxo , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Proteínas que Contêm BromodomínioRESUMO
The degradation of oncoproteins mediated by proteolysis-targeting chimera (PROTAC) has emerged as a potent strategy in cancer therapy. However, the clinical application of PROTACs is hampered by challenges such as poor water solubility and off-target adverse effects. Herein, we present an ultrasound (US)-activatable PROTAC prodrug termed NPCe6+PRO for actuating efficient sono-immunotherapy in a spatiotemporally controllable manner. Specifically, US irradiation, which exhibits deep-tissue penetration capability, results in Ce6-mediated generation of ROS, facilitating sonodynamic therapy (SDT) and inducing immunogenic cell death (ICD). Simultaneously, the generated ROS cleaves the thioketal (TK) linker through a ROS-responsive mechanism, realizing the on-demand activation of the PROTAC prodrug in deep tissues. This prodrug activation results in the degradation of the target protein BRD4, while simultaneously reversing the upregulation of PD-L1 expression associated with the SDT process. In the orthotopic mouse model of pancreatic tumors, NPCe6+PRO effectively suppressed tumor growth in conjunction with US stimulation.
Assuntos
Imunoterapia , Neoplasias Pancreáticas , Pró-Fármacos , Animais , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Pró-Fármacos/química , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/imunologia , Camundongos , Humanos , Linhagem Celular Tumoral , Proteólise/efeitos dos fármacos , Terapia por Ultrassom/métodos , Antígeno B7-H1 , Fatores de Transcrição , Proteínas de Ciclo Celular , Espécies Reativas de Oxigênio/metabolismo , Proteínas que Contêm BromodomínioRESUMO
Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
Assuntos
Proteínas de Ciclo Celular , Proteólise , Transdução de Sinais , Fatores de Transcrição , Ubiquitinação , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas que Contêm BromodomínioRESUMO
Background: Lung squamous cell carcinoma (LUSC) ranks among the carcinomas with the highest incidence and dismal survival rates, suffering from a lack of effective therapeutic strategies. Consequently, biomarkers facilitating early diagnosis of LUSC could significantly enhance patient survival. This study aims to identify novel biomarkers for LUSC. Methods: Utilizing the TCGA, GTEx, and CGGA databases, we focused on the gene encoding Family with Sequence Similarity 20, Member A (FAM20A) across various cancers. We then corroborated these bioinformatic predictions with clinical samples. A range of analytical tools, including Kaplan-Meier, MethSurv database, Wilcoxon rank-sum, Kruskal-Wallis tests, Gene Set Enrichment Analysis, and TIMER database, were employed to assess the diagnostic and prognostic value of FAM20A in LUSC. These tools also helped evaluate immune cell infiltration, immune checkpoint genes, DNA repair-related genes, DNA methylation, and tumor-related pathways. Results: FAM20A expression was found to be significantly reduced in LUSC, correlating with lower survival rates. It exhibited a negative correlation with key proteins in DNA repair signaling pathways, potentially contributing to LUSC's radiotherapy resistance. Additionally, FAM20A showed a positive correlation with immune checkpoints like CTLA-4, indicating potential heightened sensitivity to immunotherapies targeting these checkpoints. Conclusion: FAM20A emerges as a promising diagnostic and prognostic biomarker for LUSC, offering potential clinical applications.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/imunologia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Prognóstico , Regulação Neoplásica da Expressão Gênica , Biologia Computacional/métodos , Bases de Dados Genéticas , Proteínas que Contêm Bromodomínio , Proteínas do Tecido Nervoso , Fatores de Transcrição , Antígenos NuclearesRESUMO
Many inflammatory disorders, including diabetic kidney disease (DKD), are associated with pyroptosis, a type of inflammation-regulated cell death. The purpose of this work was to ascertain the effects of apabetalone, which targets BRD4, a specific inhibitor of the bromodomain (BRD) and extra-terminal (BET) proteins that target bromodomain 2, on kidney injury in DKD. This study utilized pharmacological and genetic approaches to investigate the effects of apabetalone on pyroptosis in db/db mice and human tubular epithelial cells (HK-2). BRD4 levels were elevated in HK-2 cells exposed to high glucose and in db/db mice. Modulating BRD4 levels led to changes in the generation of inflammatory cytokines and cell pyroptosis linked to NLRP3 inflammasome in HK-2 cells and db/db mice. Likewise, these cellular processes were mitigated by apabetalone through inhibition BRD4. Apabetalone or BRD4 siRNA suppressed PLK1 expression in HK-2 cells under high glucose by P300-dependent H3K27 acetylation on the PLK1 gene promoter, as demonstrated through chromatin immunoprecipitation and immunoprecipitation assays. To summarize, apabetalone relieves renal proptosis and fibrosis in DKD. BRD4 regulates the P300/H3K27ac/PLK1 axis, leading to the activation of the NLRP3 inflammasome and subsequent cell pyroptosis, inflammation, and fibrosis. These results may provide new perspectives on DKD treatment.