Novel BRD4-p53 Inhibitor SDU-071 Suppresses Proliferation and Migration of MDA-MB-231 Triple-Negative Breast Cancer Cells.

A promising alternative for cancer treatment involves targeted inhibition of the epigenetic regulator bromodomain-containing protein 4 (BRD4); however, available BRD4 inhibitors are constrained by their potency, oral bioavailability, and cytotoxicity. Herein, to overcome the drawback of the translational BRD4 inhibitors, we describe a novel BRD4-p53 inhibitor, SDU-071, which suppresses BRD4 interaction with the p53 tumor suppressor and its biological activity in MDA-MB-231 triple-negative breast cancer (TNBC) cells in vitro and in vivo. This novel small-molecule BRD4-p53 inhibitor suppresses cell proliferation, migration, and invasion by downregulating the expression of BRD4-targeted genes, such as c-Myc and Mucin 5AC, and inducing cell cycle arrest and apoptosis, as demonstrated in cultured MDA-MB-231 TNBC cells. Its antitumor activity is illustrated in an orthotopic mouse xenograft mammary tumor model. Overall, our results show that SDU-071 is a viable option for potentially treating TNBC as a new BRD4-p53 inhibitor.

BRD4 isoforms have distinct roles in tumour progression and metastasis in rhabdomyosarcoma.

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analysed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in fusion negative embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumour progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumour growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. In fusion positive alveolar rhabdomyosarcoma, BRD4-L is unrestricted in its oncogenic role, with no evident involvement of BRD4-S. Our work unveils isoform-specific functions of BRD4 in rhabdomyosarcoma.

CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation.

Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-ß activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5-/- DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5-/- DF-1 cells had increased IFN-ß production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-ß production and interferon-stimulated gene expression.

IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors.

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function.

IMPORTANCE: Human papillomavirus 16 (HPV16) is a causative agent in around 3%-4% of all human cancers, and currently, there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

The matrix protein of respiratory syncytial virus suppresses interferon signaling via RACK1 association.

IMPORTANCE: Respiratory syncytial virus (RSV) matrix (M) protein is indispensable for virion assembly and release. It is localized to the nucleus during early infection to perturb host transcription. However, the function of RSV M protein in other cellular activities remains poorly understood. In this study, several interferon response-associated host factors, including RACK1, were identified by proteomic analysis as RSV M interactors. Knockdown of RACK1 attenuates RSV-restricted IFN signaling leading to enhanced host defense against RSV infection, unraveling a role of M protein in antagonizing IFN response via association with RACK1. Our study uncovers a previously unrecognized mechanism of immune evasion by RSV M protein and identifies RACK1 as a novel host factor recruited by RSV, highlighting RACK1 as a potential new target for RSV therapeutics development.

BRD4 isoforms have distinct roles in tumor progression and metastasis in embryonal rhabdomyosarcoma.

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analyzed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumor progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumor growth, but strikingly promotes metastasis in vivo . Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. Our work unveils isoform-specific functions of BRD4 and demonstrates that BRD4-S functions as a gatekeeper to constrain the full oncogenic potential of BRD4-L.

Erratum: N6-methyladenosine-induced ERRγ triggers chemoresistance of cancer cells through upregulation of ABCB1 and metabolic reprogramming: Erratum.

[This corrects the article DOI: 10.7150/thno.40144.].

Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function.

During the human papillomavirus 16 life cycle, the E2 protein binds simultaneously to the viral genome and host chromatin throughout mitosis, ensuring viral genomes reside in daughter cell nuclei following cell division. Previously, we demonstrated that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, and that this interaction is required for optimum E2 mitotic chromatin association and plasmid segregation function. Others have implicated BRD4 in mediating the plasmid segregation function of E2 and we have demonstrated that there is a TopBP1-BRD4 complex in the cell. We therefore further investigated the role of the E2-BRD4 interaction in mediating E2 association with mitotic chromatin and plasmid segregation function. Using a combination of immunofluorescence and our novel plasmid segregation assay in U2OS and N/Tert-1 cells stably expressing a variety of E2 mutants, we report that direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for E2 association with mitotic chromatin and plasmid segregation. We also identify a novel TopBP1 mediated interaction between E2 and the BRD4 extra-terminal (ET) domain in vivo . Overall, the results demonstrate that direct interaction with TopBP1 and the BRD4 CTM are required for E2 mitotic chromatin association and plasmid segregation function. Disruption of this complex offers therapeutic options for targeting segregation of viral genomes into daughter cells, potentially combatting HPV16 infections, and cancers that retain episomal genomes. Importance: HPV16 is a causative agent in around 3-4% of all human cancers and currently there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

MYC promotes global transcription in part by controlling P-TEFb complex formation via DNA-binding independent inhibition of CDK9 SUMOylation.

MYC is an oncogenic transcription factor with a novel role in enhancing global transcription when overexpressed. However, how MYC promotes global transcription remains controversial. Here, we used a series of MYC mutants to dissect the molecular basis for MYC-driven global transcription. We found that MYC mutants deficient in DNA binding or known transcriptional activation activities can still promote global transcription and enhance serine 2 phosphorylation (Ser2P) of the RNA polymerase (Pol) II C-terminal domain (CTD), a hallmark of active elongating RNA Pol II. Two distinct regions within MYC can promote global transcription and Ser2P of Pol II CTD. The ability of various MYC mutants to promote global transcription and Ser2P correlates with their ability to suppress CDK9 SUMOylation and enhance positive transcription elongation factor b (P-TEFb) complex formation. We showed that MYC suppresses CDK9 SUMOylation by inhibiting the interaction between CDK9 and SUMO enzymes including UBC9 and PIAS1. Furthermore, MYC's activity in enhancing global transcription positively contributes to its activity in promoting cell proliferation and transformation. Together, our study demonstrates that MYC promotes global transcription, at least in part, by promoting the formation of the active P-TEFb complex via a sequence-specific DNA-binding activity-independent manner.

EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration-approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.

Conditional Human BRD4 Knock-In Transgenic Mouse Genotyping and Protein Isoform Detection.

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions. Graphic abstract.

Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site.

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.

Dual-target inhibitors of bromodomain and extra-terminal proteins in cancer: A review from medicinal chemistry perspectives.

Bromodomain-containing protein 4 (BRD4), as the most studied member of the bromodomain and extra-terminal (BET) family, is a chromatin reader protein interpreting epigenetic codes through binding to acetylated histones and non-histone proteins, thereby regulating diverse cellular processes including cell cycle, cell differentiation, and cell proliferation. As a promising drug target, BRD4 function is closely related to cancer, inflammation, cardiovascular disease, and liver fibrosis. Currently, clinical resistance to BET inhibitors has limited their applications but synergistic antitumor effects have been observed when used in combination with other tumor inhibitors targeting additional cellular components such as PLK1, HDAC, CDK, and PARP1. Therefore, designing dual-target inhibitors of BET bromodomains is a rational strategy in cancer treatment to increase potency and reduce drug resistance. This review summarizes the protein structures and biological functions of BRD4 and discusses recent advances of dual BET inhibitors from a medicinal chemistry perspective. We also discuss the current design and discovery strategies for dual BET inhibitors, providing insight into potential discovery of additional dual-target BET inhibitors.

Discovery of Novel Dual-Target Inhibitor of Bromodomain-Containing Protein 4/Casein Kinase 2 Inducing Apoptosis and Autophagy-Associated Cell Death for Triple-Negative Breast Cancer Therapy.

Bromodomain-containing protein 4 (BRD4) is an attractive epigenetic target in human cancers. Inhibiting the phosphorylation of BRD4 by casein kinase 2 (CK2) is a potential strategy to overcome drug resistance in cancer therapy. The present study describes the synthesis of multiple BRD4-CK2 dual inhibitors based on rational drug design, structure-activity relationship, and in vitro and in vivo evaluations, and 44e was identified to possess potent and balanced activities against BRD4 (IC50 = 180 nM) and CK2 (IC50 = 230 nM). In vitro experiments show that 44e could inhibit the proliferation and induce apoptosis and autophagy-associated cell death of MDA-MB-231 and MDA-MB-468 cells. In two in vivo xenograft mouse models, 44e displays potent anticancer activity without obvious toxicities. Taken together, we successfully synthesized the first highly effective BRD4-CK2 dual inhibitor, which is expected to be an attractive therapeutic strategy for triple-negative breast cancer (TNBC).

Functional coordination of BET family proteins underlies altered transcription associated with memory impairment in fragile X syndrome.

Bromodomain and extraterminal proteins (BET) are epigenetic readers that play critical roles in gene regulation. Pharmacologic inhibition of the bromodomain present in all BET family members is a promising therapeutic strategy for various diseases, but its impact on individual family members has not been well understood. Using a transcriptional induction paradigm in neurons, we have systematically demonstrated that three major BET family proteins (BRD2/3/4) participated in transcription with different recruitment kinetics, interdependency, and sensitivity to a bromodomain inhibitor, JQ1. In a mouse model of fragile X syndrome (FXS), BRD2/3 and BRD4 showed oppositely altered expression and chromatin binding, correlating with transcriptional dysregulation. Acute inhibition of CBP/p300 histone acetyltransferase (HAT) activity restored the altered binding patterns of BRD2 and BRD4 and rescued memory impairment in FXS. Our study emphasizes the importance of understanding the BET coordination controlled by a balanced action between HATs with different substrate specificity.

Expression of Phosphorylated BRD4 Is Markedly Associated with the Activation Status of the PP2A Pathway and Shows a Strong Prognostic Value in Triple Negative Breast Cancer Patients.

The bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, has emerged in the last years as a promising molecular target in many tumors including breast cancer. The triple negative breast cancer (TNBC) represents the molecular subtype with the worst prognosis and a current therapeutic challenge, and TNBC cells have been reported to show a preferential sensitivity to BET inhibitors. Interestingly, BRD4 phosphorylation (pBRD4) was found as an alteration that confers resistance to BET inhibition and PP2A proposed as the phosphatase responsible to regulate pBRD4 levels. However, the potential clinical significance of pBRD4, as well as its potential correlation with the PP2A pathway in TNBC, remains to be investigated. Here, we evaluated the expression levels of pBRD4 in a series of 132 TNBC patients. We found high pBRD4 levels in 34.1% of cases (45/132), and this alteration was found to be associated with the development of patient recurrences (p = 0.007). Interestingly, BRD4 hyperphosphorylation predicted significantly shorter overall (p < 0.001) and event-free survival (p < 0.001). Moreover, multivariate analyses were performed to confirm its independent prognostic impact in our cohort. In conclusion, our findings show that BRD4 hyperphosphorylation is an alteration associated with PP2A inhibition that defines a subgroup of TNBC patients with unfavorable prognosis, suggesting the potential clinical and therapeutic usefulness of the PP2A/BRD4 axis as a novel molecular target to overcome resistance to treatments based on BRD4 inhibition.