Transcriptional control of gene expression is fundamental to all cellular processes. Conversely, transcriptional dysregulation is a hallmark of cancer. While this hallmark is a key driver of all malignancy-related process, it also represents a vulnerability that can be exploited therapeutically. Prostate cancer is a prime example of this phenomenon: it is characterised by aberrant transcription and treated with drugs that influence transcriptional pathways. Indeed, the primary oncogenic driver and therapeutic target of prostate cancer, the androgen receptor (AR), is a transcription factor. Moreover, a plethora of other transcriptional regulators, including transcriptional cyclin-dependent kinases (CDK7, CDK8 and CDK9), MYC and Bromodomain-containing protein 4 (BRD4), play prominent roles in disease progression. In this review, we focus on the roles of transcriptional CDKs in prostate cancer growth, metastasis and therapy resistance and discuss their interplay with AR, MYC and BRD4. Additionally, we explore recent advances in the therapeutic targeting of transcriptional CDKs and propose how these strategies could be effectively harnessed for the treatment of prostate cancer.

BET bromodomain inhibitors demonstrate significant promise as anti-inflammatory agents. However, clinical data demonstrated that nonselective BET bromodomain inhibitors led to significant dose-limiting toxicity in clinical settings. Here, we use three orally bioavailable inhibitors, 1-3, that are either BRD4-D1 selective or pan-D1-biased + BRD4-D2, for assessing their cellular and in vivo efficacy and safety profile compared to known BET inhibitors in two inflammatory disease models. Our results show that pan-D1-biased + BRD4-D2 inhibitor, 3, is as efficacious as pan-BET inhibitor, I-BET151, in reducing inflammation in both models, whereas pan-D2 inhibitors are less effective. BRD4-D1 selective inhibitors are also efficacious; however, inhibitors with improved cellular engagement will be necessary to better assess their effects. Finally, BRD4-D1 selective inhibitors are better tolerated in a preclinical thrombocytopenia model than 3, while gastrointestinal toxicity may be a BRD4-driven effect. These results highlight the importance of assessing specific BET bromodomain functions due to their diverse roles in disease models.

Recent work by the ICGC-PCAWG consortium identified recurrent focal deletions in the BRD4 gene, decreasing expression despite increased copy number. We show that these focal deletions occur in the context of cyclin E1 amplification in breast, ovarian, and endometrial cancers, and serve to disrupt BRD4 regulatory regions and gene expression across isoforms. We analyze open reading frame screen data and find that overexpression of BRD4 long (BRD4-L) and short isoform BRD4-S(a) impairs cell growth across cell lines. We confirm these results in OVSAHO ovarian cancer cells, where the overexpression of BRD4 isoforms significantly reduces tumor growth. Next, we mimic BRD4 focal deletions using CRISPR-Cas9 technology and show that these focal deletions rescue ovarian cancer cells from toxicity associated with BRD4 overexpression, suggesting that BRD4 levels must be fine-tuned for cancer cell proliferation. Our study provides experimental evidence for the first recurrent deletion reducing toxicity in cancer, expanding the landscape of cancer progression mechanisms.

Promoter-promoter and enhancer-promoter interactions are enriched in histone acetylation and central to chromatin organization in active genetic regions. Bromodomains are epigenetic "readers" that recognize and bind histone acetylation. Bromodomains often exist in tandem or with other reader domains. Cellular knockdown of the bromodomain and extraterminal domain (BET) protein family disrupts chromatin organization, but the mechanisms through which BET proteins preserve chromatin structure are largely unknown. We hypothesize that BET proteins maintain overall chromatin structure by employing their tandem bromodomains to multivalently scaffold acetylated nucleosomes in an intranucleosomal or internucleosomal manner. To test this hypothesis biophysically, we used small-angle X-ray scattering, electron paramagnetic resonance, and Rosetta protein modeling to show that a disordered linker separates BET tandem bromodomain acetylation binding sites by 15 to 157 Å. Most of these modeled distances are sufficient to span the length of a nucleosome (>57 Å). Focusing on the BET family member BRD4, we employed bioluminescence resonance energy transfer and isothermal titration calorimetry to show that BRD4 bromodomain binding of multiple acetylation sites on a histone tail does not increase BRD4-histone tail affinity, suggesting that BET bromodomain intranucleosome binding is not biologically relevant. Using sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays, we provide the first direct biophysical evidence that BET bromodomains can scaffold multiple acetylated nucleosomes. Taken together, our results demonstrate that BET bromodomains are capable of multivalent internucleosome scaffolding in vitro. The knowledge gained provides implications for how BET bromodomain-mediated acetylated internucleosome scaffolding may maintain cellular chromatin interactions in active genetic regions.

The bromodomain and extraterminal domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor because of an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, bromodomain-containing protein 4 (BRD4) binds to estrogen receptor binding sites and activates transcription of critical oncogenes such as MYC, independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator reduces BRD4's enhancer occupancy. Profiling changes of the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6 and the polymerase-associated factor 1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies.

This study aims to find whether the proliferation and migration of triple negative breast cancer (TNBC) cell lines can be reduced by treatment with bromodomain and extra-terminal domain (BET) inhibitor JQ1 and BET protein targeting chimeras (PROTACs) ARV-771 and MZ1.

Cytotoxicity tests, scratch migration assays and western blot proteome profiler arrays for protein expression of cancer-related proteins were used to evaluate the impact of a BET-inhibitor and two BET-directed PROTACs on cell viability, migration and on protein expression.

JQ1 and the PROTACs MZ1 and ARV-771 significantly inhibited the growth and migration of the KRAS G13D-mutated MDA-MB-231 cells. In this cell line, the PROTACs suppressed the residual expression of ERBB2/HER2, 3 and 4 that are essential for the proliferation of breast cancer cells and this cell line proved sensitive to HER2 inhibitors. In contrast, the effects of the PROTACs on the protein expression of MDA-MB-436 cells mostly affected cytokines and their cognate receptors.

The degradation of BET-protein by PROTACs demonstrated significant anti-proliferative effects. The KRAS-mutated MDA-MB-231 cells belong to the low-HER2 expressing tumors that have a poorer prognosis compared to HER2-null patients. Since first oral PROTACs against tumor hormone receptors are in clinical trials, this mode of tumor therapy is expected to become an important therapeutic strategy in the future treatment of TNBC.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder, mainly comprising two subtypes: ulcerative colitis (UC) and Crohn's disease (CD). IBD, featured by recurrent symptoms and significant morbidity, poses a significant threat to global health and has an adverse impact on quality of life. Currently, there is no curative therapy for IBD, and the available medications are only for managing the disease condition, likely owing to the insufficient understanding of the underlying pathophysiology processes involved in IBD, and the lack of safe and effective medicines. Thus, novel targeted therapies for IBD are urgently needed for better efficacy with an improved adverse event profile. As the most extensively studied member of bromodomain and extra terminal domain (BET) family proteins, bromodomain-containing protein 4 (BRD4) is emerging as a promising epigenetic therapeutic target for IBD. Pharmacological inhibition of BRD4 with selective small molecule inhibitors shows potent anti-inflammatory effects in both in vitro and different IBD mouse models. Herein, we summarize current knowledge in understanding the role of BRD4 in the pathogenesis and development of IBD, and the clinical landscape of developing BET/BRD4 inhibitors and emerging BRD4-targeted degraders as promising therapeutical alternatives. Challenges and opportunities, as well as future directions in drug discovery by targeting BRD4 are also briefly discussed.

R-loops are triple-stranded nucleic acid structures that occur when newly synthesized single-stranded RNA anneals to duplex DNA upon the collision of replication forks with transcription complexes. These RNA-DNA hybrids facilitate several transcriptional processes in the cell and have been described extensively in the literature. Recently, evidence has emerged that R-loops are key regulators of DNA tumor virus transcription and the replication of their lifecycle. Studies have demonstrated that R-loops on the Human Papillomavirus (HPV) genome must be resolved to maintain genome maintenance and avoid viral integration, a hallmark of HPV cancers. For Epstein-Barr virus (EBV), R-loops are formed at the oriLyt to establish lytic replication. Structural maintenance of chromosome proteins 5/6 (SMC5/6) bind to these viral R-loops to repress EBV lytic replication. Most viruses in the herpesvirales order, such as KSHV, contain R-loop-forming sequences. In this perspective, we will describe the current, although limited, literature demonstrating the importance of RNA-DNA hybrids to regulate DNA virus transcription. We will also detail potential new areas of R-loop research and how these viruses can be used as tools to study the growing field of R-loops.

Bromodomain-containing protein 4 (BRD4), an important epigenetic reader, is closely associated with the pathogenesis and development of many diseases, including various cancers, inflammation, and infectious diseases. Targeting BRD4 inhibition or protein elimination with small molecules represents a promising therapeutic strategy, particularly for cancer therapy.

The recent advances of patented BRD4 degraders were summarized. The challenges, opportunities, and future directions for developing novel potent and selective BRD4 degraders are also discussed. The patents of BRD4 degraders were searched using the SciFinder and Cortellis Drug Discovery Intelligence database.

BRD4 degraders exhibit superior efficacy and selectivity to BRD4 inhibitors, given their unique mechanism of protein degradation instead of protein inhibition. Excitingly, RNK05047 is now in phase I/II clinical trials, indicating that selective BRD4 protein degradation may offer a viable therapeutic strategy, particularly for cancer. Targeting BRD4 with small-molecule degraders provides a promising approach with the potential to overcome therapeutic resistance for treating various BRD4-associated diseases.

Lower limb arterial occlusive disease is treated with intraluminal devices, such as paclitaxel (PTX)-coated balloons (PCBs); however, post-procedural restenosis remains a significant challenge. NLRP3 activation is known to play a significant role in atherosclerosis, but its involvement in restenosis following PCB intervention remains to be investigated. We identified that NLRP3 was differentially expressed in lower-limb arterial tissues sourced from healthy controls and patients with arterial occlusive disease. Through cell experiments, we confirmed that PTX is involved in the activation of NLRP3. Subsequently, we demonstrated that NLRP3 activation promotes the proliferation and migration of vascular smooth muscle cell (VSMC), thereby reducing their sensitivity to PTX. NLRP3 activation also stimulates the secretion of the inflammatory cytokine interleukin IL-1β. RNA sequencing of IL-1β-treated VSMC revealed the upregulation of BRD4 and LIN9. Further mechanistic investigations confirmed that IL-1β facilitates BRD4 recruitment, leading to enhanced LIN9 expression. The transcription factor LIN9 binds to the promoter region of the cell-cycle regulator AURKA, thereby promoting its transcription and subsequently upregulating the expression of the cell proliferation-associated molecule FOXM1. These processes ultimately mediate the proliferation, migration, and PTX resistance of VSMC. Additionally, we discovered that JQ1 inhibited the overexpression of the above molecules, and exhibited a synergistic effect with PTX. Our conclusions were validated through in vivo experiments in Sprague-Dawley rats. Collectively, our findings provide insights into the molecular mechanisms underlying restenosis following PCB therapy, and suggest that the combined use of JQ1 and PTX devices may represent a promising therapeutic strategy.

iPSC reprogramming technology exhibits significant promise in the realms of clinical therapeutics, disease modeling, pharmaceutical drug discovery, and various other applications. However, the extensive utilization of this technology has encountered impediments in the form of inefficiency, prolonged procedures, and ambiguous biological processes. Consequently, in order to improve this technology, it is of great significance to delve into the underlying mechanisms involved in iPSC reprogramming. The BET protein BRD4 plays a crucial role in the late stage of reprogramming; however, its precise function in the early stage remains unclear.

Our study aims to investigate BRD4's role in the early stages of iPSC reprogramming. Our investigation reveals that early inhibition of BRD4 substantially enhances iPSC reprogramming, whereas its implementation during the middle-late stage impedes the process. During the reprogramming, ribosome DNA expression initially increases before decreasing and then gradually recovers. Early inhibition of BRD4 improved the decline and restoration of rDNA expression in the early and middle-late stages, respectively. Additionally, we uncovered the mechanism of BRD4's regulation of rDNA transcription throughout reprogramming. Specifically, BRD4 interacts with UBF and co-localizes to both the rDNA promoter and enhancer regions. Ultimately, BRD4 facilitates rDNA transcription by promoting the enrichment of histone H3 lysine 27 acetylation in the surrounding chromatin. Moreover, we also discovered that early inhibition of BRD4 facilitates cells' transition out of the somatic cell state and activate pluripotent genes.

In conclusion, our results demonstrate that early inhibition of BRD4 promotes sequential dynamic expression of rDNA, which improves iPSC reprogramming efficiency.

Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Fibrosis is an excessive wound-healing response induced by repeated or chronic external stimuli to tissues, significantly impacting quality of life and primarily contributing to organ failure. Organ fibrosis is reported to cause 45% of all-cause mortality worldwide. Despite extensive efforts to develop new antifibrotic drugs, drug discovery has not kept pace with the clinical demand. Currently, only pirfenidone and nintedanib are approved by the FDA to treat pulmonary fibrotic illness, whereas there are currently no available antifibrotic drugs for hepatic, cardiac or renal fibrosis. The development of fibrosis is closely related to epigenetic alterations. The field of epigenetics primarily studies biological processes, including chromatin modifications, epigenetic readers, DNA transcription and RNA translation. The bromodomain and extra-terminal structural domain (BET) family, a class of epigenetic readers, specifically recognizes acetylated histone lysine residues and promotes the formation of transcriptional complexes. Bromodomain-containing protein 4 (BRD4) is one of the most well-researched proteins in the BET family. BRD4 is implicated in the expression of genes related to inflammation and pro-fibrosis during fibrosis. Inhibition of BRD4 has shown promising anti-fibrotic effects in preclinical studies; however, no BRD4 inhibitor has been approved for clinical use. This review introduces the structure and function of BET proteins, the research progress on BRD4 in organ fibrosis, and the inhibitors of BRD4 utilized in fibrosis. We emphasize the feasibility of targeting BRD4 as an anti-fibrotic strategy and discuss the therapeutic potential and challenges associated with BRD4 inhibitors in treating fibrotic diseases.

The senescent microenvironment and aged cells per se contribute to tissue remodeling, chronic inflammation, and age-associated dysfunction. However, the metabolic and epigenomic bases of the senescence-associated secretory phenotype (SASP) remain largely unknown. Here, we show that ATP-citrate lyase (ACLY), a key enzyme in acetyl-coenzyme A (CoA) synthesis, is essential for the pro-inflammatory SASP, independent of persistent growth arrest in senescent cells. Citrate-derived acetyl-CoA facilitates the action of SASP gene enhancers. ACLY-dependent de novo enhancers augment the recruitment of the chromatin reader BRD4, which causes SASP activation. Consistently, specific inhibitions of the ACLY-BRD4 axis suppress the STAT1-mediated interferon response, creating the pro-inflammatory microenvironment in senescent cells and tissues. Our results demonstrate that ACLY-dependent citrate metabolism represents a selective target for controlling SASP designed to promote healthy aging.

The Bromodomain and Extra-Terminal Domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor due to an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, BRD4 binds to estrogen receptor binding sites and activates transcription of critical oncogenes independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator complex reduces BRD4's enhancer occupancy. Profiling changes in the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6, and PAF1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies.

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.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

As a member of BET (bromodomain and extra-terminal) protein family, BRD4 (bromodomain‑containing protein 4) is a chromatin‑associated protein that interacts with acetylated histones and actively recruits regulatory proteins, leading to the modulation of gene expression and chromatin remodeling. The cellular and epigenetic functions of BRD4 implicate normal development, fibrosis and inflammation. BRD4 has been suggested as a potential therapeutic target as it is often overexpressed and plays a critical role in regulating gene expression programs that drive tumor cell proliferation, survival, migration and drug resistance. To address the roles of BRD4 in cancer, several drugs that specifically target BRD4 have been developed. Inhibition of BRD4 has shown promising results in preclinical models, with several BRD4 inhibitors undergoing clinical trials for the treatment of various cancers. Head and neck squamous cell carcinoma (HNSCC), a heterogeneous group of cancers, remains a health challenge with a high incidence rate and poor prognosis. Conventional therapies for HNSCC often cause adverse effects to the patients. Targeting BRD4, therefore, represents a promising strategy to sensitize HNSCC to chemo‑ and radiotherapy allowing de‑intensification of the current therapeutic regime and subsequent reduced side effects. However, further studies are required to fully understand the underlying mechanisms of action of BRD4 in HNSCC in order to determine the optimal dosing and administration of BRD4‑targeted drugs for the treatment of patients with HNSCC.

Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Decades of scientific research have been devoted to unraveling the intricacies of eukaryotic transcription since the groundbreaking discovery of eukaryotic RNA polymerases in the late 1960s. RNA polymerase II, the polymerase responsible for mRNA synthesis, has always attracted the most attention. Despite its structural resemblance to its bacterial counterpart, eukaryotic RNA polymerase II faces a unique challenge in progressing transcription due to the presence of nucleosomes that package DNA in the nuclei. In this review, we delve into the impact of RNA polymerase II and histone signaling on the progression of eukaryotic transcription. We explore the pivotal points of interactions that bridge the RNA polymerase II and histone signaling systems. Finally, we present an analysis of recent cryo-electron microscopy structures, which captured RNA polymerase II-nucleosome complexes at different stages of the transcription cycle. The combination of the signaling crosstalk and the direct visualization of RNA polymerase II-nucleosome complexes provides a deeper understanding of the communication between these two major players in eukaryotic transcription.

Aberrant expression of critical components of the trans-acting super-enhancers (SE) complex contributes to the continuous and robust transcription of oncogenes in human cancers. Small-molecule inhibitors targeting core-transcriptional components such as transcriptional bromodomain protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7) have been developed and are currently undergoing preclinical and clinical testing in several malignant cancers. By analysis of TCGA data and clinical specimens, we demonstrated that BRD4 and CDK7 were frequently overexpressed in human HCCs and were associated with the poor prognosis. Shorter survival and poorly differentiated histology were linked to high BRD4 or CDK7 expression levels. Interestingly, co-overexpression of BRD4 and CDK7 was a more unfavorable prognostic factor in HCC. Treatment with JQ1 or THZ1 alone exhibited an inhibitory impact on the proliferation of HCC cells, while JQ1 synergized with THZ1 showed a more pronounced suppression. Concurrently, a combined JQ1 and THZ1 treatment abolished the transcription of oncogenes ETV4, MYC, NFE2L2. Our study suggested that BRD4 and CDK7 coupled can be a valuable biomarker in HCC diagnosis and the combination of JQ1 and THZ1 can be a promising therapeutic treatment against HCC.

Bromodomain-containing protein 4 (BRD4) inhibitors have been proven to be a promising option for anti-HIV-1 latency therapeutics. We herein describe the design, synthesis, and anti-HIV-1 latency bioevaluation of triazolopyridine derivatives as BRD4 inhibitors. Among them, compound 13d displayed favorable HIV-1 reactivation and prominent safety profile without triggering abnormal immune activation. It exerted strong synergism when combined with the PKC activator prostratin and has the same BRD4-targeting latency mechanism as observed with JQ1, by stimulating Tat-dependent HIV-1 elongation. Besides, it neither affected the antiviral efficacies of antiviral drugs nor caused secondary infections to uninfected cells and the latency reversing potency of 13d, in turn, was not affected by different classes of antiviral drugs.

Ovarian cancer is one of the most lethal gynecological cancers worldwide, with approximately 70% of cases diagnosed in advanced stages. This late diagnosis results from the absence of early warning symptoms and is associated with an unfavorable prognosis. A standard treatment entails a combination of primary chemotherapy with platinum and taxane agents. Tumor recurrence following first-line chemotherapy with Carboplatin and Paclitaxel is detected in 80% of advanced ovarian cancer patients, with disease relapse occurring within 2 years of initial treatment. Platinum-resistant ovarian cancer is one of the biggest challenges in treating patients. Second-line treatments involve PARP or VEGF inhibitors. Identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have determined that low Caspase-8 expression in ovarian cancer patients leads to poor prognosis. High-Grade Serous Ovarian Cancer (HGSOC) cells lacking Caspase-8 expression showed an altered composition of the RNA Polymerase II-containing transcriptional elongation complex leading to increased transcriptional activity. Caspase-8 knockout cells display increased BRD4 expression and CDK9 activity and reduced sensitivities to Carboplatin and Paclitaxel. Based on our work, we are proposing three potential therapeutic approaches to treat advanced ovarian cancer patients who exhibit low Caspase-8 expression and resistance to Carboplatin and/or Paclitaxel-combinations of (1) Carboplatin with small-molecule BRD4 inhibitors; (2) Paclitaxel with small-molecule BRD4 inhibitors, and (3) small-molecule BRD4 and CDK9 inhibitors. In addition, we are also proposing two predictive markers of chemoresistance-BRD4 and pCDK9.

Antiretroviral therapy (ART) has brought the HIV/AIDS epidemic under control, but a curative strategy for viral eradication is still needed. The cessation of ART results in rapid viral rebound from latently infected CD4+ T cells, showing that control of viral replication alone does not fully restore immune function, nor does it eradicate viral reservoirs. With a better understanding of factors and mechanisms that promote viral latency, current approaches are primarily focused on the permanent silencing of latently infected cells ("block and lock") or reactivating HIV-1 gene expression in latently infected cells, in combination with immune restoration strategies to eliminate HIV infected cells from the host ("shock and kill"). In this review, we provide a summary of the current, most promising approaches for HIV-1 cure strategies, including an analysis of both latency-promoting agents (LPA) and latency-reversing agents (LRA) that have shown promise in vitro, ex vivo, and in human clinical trials to reduce the HIV-1 reservoir.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

Epigenetic changes contribute to the profound alteration in the transcriptional program associated with the onset and progression of muscle wasting in several pathological conditions. Although HDACs and their inhibitors have been extensively studied in the field of muscular dystrophies, the potential of epigenetic inhibitors has only been marginally explored in other disorders associated with muscle atrophy, such as in cancer cachexia and sarcopenia. BET inhibitors represent a novel class of recently developed epigenetic drugs that display beneficial effects in a variety of diseases beyond malignancies. Based on the preliminary in vitro and preclinical data, HDACs and BET proteins contribute to the pathogenesis of cancer cachexia and sarcopenia, modulating processes related to skeletal muscle mass maintenance and/or metabolism. Thus, epigenetic drugs targeting HDACs and BET proteins may emerge as promising strategies to reverse the catabolic phenotype associated with cachexia and sarcopenia. Further preclinical studies are warranted to delve deeper into the molecular mechanisms associated with the functions of HDACs and BET proteins in muscle atrophy and to establish whether their epigenetic inhibitors represent a prospective therapeutic avenue to alleviate muscle wasting.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

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.

Aberrant interplay between epigenetic reprogramming and hypoxia signaling contributes to renal cell carcinoma progression and drug resistance, which is an essential hallmark. How the chromatin remodelers enhance RCC malignancy remains to be poorly understood. We aimed to elucidate the roles of CHD1L in determining hypoxia signaling activation and sunitinib resistance.

The qRT-PCR, western blotting, and immunohistochemistry technologies were used to detect CHD1L expressions. Lentivirus transfection was used to generate stable CHD1L-KD cells. The roles of SIRT7/CHD1L were evaluated by CCK-8, wound healing, transwell assays, xenograft models, and tail-vein metastasis models. Co-immunoprecipitation, Chromatin Immunoprecipitation (ChIP), and luciferase reporter assays were conducted to explore epigenetic regulations.

We screened and validated that CHD1L is up-regulated in RCC and correlates with poorer prognosis of patients. CHD1L overexpression notably enhances cell proliferation, migration, and self-renewal capacities in vitro and in vivo. Mechanistically, SIRT7 physically interacts with CHDL1 and mediates the deacetylation of CHD1L. Wild-type SIRT7, but not H187Y dead mutant, stabilizes CHD1L protein levels via attenuating its ubiquitination levels. SIRT7 is increased in RCC and correlates with hazardous RCC clinical characteristics. SIRT7 depends on CHD1L to exert its tumor-promoting functions. Accumulated CHD1L amplifies HIF-2α-driven transcriptional programs via interacting with HIF-2α. CHD1L recruits BRD4 and increases the RNA polymerase II S2P loading. CHD1L ablation notably abolishes HIF-2α binding and subsequent transcriptional activation. CHD1L overexpression mediates the sunitinib resistance via sustaining VEGFA and targeting CHD1L reverses this effect. Specific CHD1L inhibitor (CHD1Li) shows a synergistic effect with sunitinib and strengthens its pharmaceutical effect.

These results uncover a CHD1L-mediated epigenetic mechanism of HIF-2α activation and downstream sunitinib resistance. The SIRT7-CHD1L-HIF-2α axis is highlighted to predict RCC prognosis and endows potential targets.

The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.

Enhancers are essential cis-acting regulatory elements that determine cell identity and tumor progression. Enhancer function is dependent on the physical interaction between the enhancer and its target promoter inside its local chromatin environment. Enhancer reprogramming is an important mechanism in cancer pathogenesis and can be driven by both cis and trans factors. Super enhancers are acquired at oncogenes in numerous cancer types and represent potential targets for cancer treatment. BET and CDK inhibitors act through mechanisms of enhancer function and have shown promising results in therapy for various types of cancer. Genome editing is another way to reprogram enhancers in cancer treatment. The relationship between enhancers and cancer has been revised by several authors in the past few years, which mainly focuses on the mechanisms by which enhancers can impact cancer. Here, we emphasize SE's role in cancer pathogenesis and the new therapies involving epigenetic regulators (BETi and CDKi). We suggest that understanding mechanisms of activity would aid clinical success for these anti-cancer agents.

Aging is a key independent risk factor of various vascular diseases, for which the regulatory mechanisms remain largely unknown. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain and Extra-Terminal domain (BET) family and is an epigenetic reader playing diverse roles in regulating transcriptional elongation, chromatin remodeling, DNA damage response, and alternative splicing in various cells and tissues. While BRD4 was initially recognized for its involvement in cancer progression, recent studies have revealed that the aberrant expression and impaired function of BRD4 were highly associated with aging-related vascular pathology, affecting multiple key biological processes in the vascular cells and tissues, providing new insights into the understanding of vascular pathophysiology and pathogenesis of vascular diseases. This review summarizes the recent advances in BRD4 biological function, and the progression of the studies related to BRD4 in aging-associated vascular pathologies and diseases, including atherosclerosis, aortic aneurism vascular neointima formation, pulmonary hypertension, and essential hypertension, providing updated information to advance our understanding of the epigenetic mechanisms in vascular diseases during aging and paving the way for future research and therapeutic approaches.