BRD4 as a potential target for HPV associated cancer

Abstract

Around 99% of cervical cancer and 5%−10% of human cancer are associated with Human Papillomaviruses (HPV). Notably, the life-cycle of HPV begins by low-level infection of the basal cells of the stratified epithelium, where the viral genomes are replicated and passed on to the daughter proliferating basal cells. The production of new viral particles remains restricted to eventually differentiated cells. HPVs support their persistent infectious cycle by hijacking pivotal pathways and cellular processes. Bromodomain-containing protein 4 (BRD4) is one of the essential cellular factors involved in multiple stages of viral transcription and replication. In this review, we demonstrate the role of BRD4 in the multiple stages of HPV infectious cycle. Also, we provide an overview of the intense research about the cellular functions of BRD4, the mechanism of action of bromodomain and extra terminal (BET) inhibitors, and how it could lead to the development of anti-viral/anti-cancer therapies.

1. Human papillomavirus (HPV) and -associated cancer

There are more than 100 different types of HPVs, which may be divided into cutaneous or mucosal, able to infect the skin of the upper-respiratory or genital tracts or mucosa tissue, respectively (1).

HPVs include a diverse group of viruses that are linked to both benign and malignant cancers. They drive the most cervical cancers, in addition to many other head and neck and anogenital cancers (2). Cervical cancer ranks second among women between the ages of 15 and 44 and fourth among women worldwide (3). E6 and E7 are the main viral oncoproteins in high-risk HPV (HR-HPV, that causes cancer) that target the tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), of the infected cell, promoting their replication and allowing them to migrate through the differentiating epithelium (4).

HPV is believed to infect the host through contact with mucous membrane, infected genital skin, body fluids, and by sexual transmission. The host immune system clears the majority (70–90%) of the infection; however, a chronic infection develops if the immune system fails to clear the virus in 5–10% of infected people and may eventually result in cancer. Chronic infection results in accidental integration of the viral episomes into the host DNA (5). This unpleasant occurrence not only prevents the virus from finishing its productive life-cycle but also encourages carcinogenesis in the host (6). In papillomaviruses (PVs), viral replication is coordinated by two viral proteins (i.e., E1 and E2) that interact with the host cellular factors to replicate the viral genome. The E2 protein forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the viral origin of replication in the long control region and recruits the viral helicase E1 to the origin of replication (7, 8). E1 interacts with host polymerases, single-stranded binding proteins and other factors involved in DNA replication (9–13), while E2 also interacts with BRD4, it also engages with other cellular proteins, such as TopBP1 and ChlR1 to facilitate the augmentation of viral replication (14–19). PVs maintain their lifestyle by hijacking and manipulating the host factors (20).

2. HIV and cancer

The increased susceptibility to cancer among individuals with human immunodeficiency virus (HIV) may be attributed to the long-term impact of certain oncogenic infections. The degree of immunological insufficiency is correlated with it. It may also have a tight association with the progression of HIV replication (21). Therefore, it may be inferred that if HIV treatment is able to achieve long-lasting HIV suppression and sustain immune function at levels close to those seen in individuals without HIV, the heightened risk of cancer is expected to be mitigated. The extent to which HIV-associated cancer risk may be minimized by achieving a level of immune function that closely approximates normal is uncertain, despite the significant reduction in cancer risk that can be achieved via effective avoidance of profound immunity. For most cancers, it is appropriate to follow general population guidelines on cancer screening. In HIV-infected children, combined antiretroviral treatment (cART) lowers the chance of getting cancer (22). People living with HIV have an increased relative risk of developing non-Hodgkin’s lymphoma (NHL) (23).

3. BRD4 regulates HPV life-cycle at multistep

One of the post-translational modifications that occurs most often in proteins is called lysine acetylation (Kac). There are “reader” proteins, which contain bromodomains (BDs), and are able to recognize acetylated proteins. Among the 46 BD-containing proteins found in humans, BRD2, BRD3, BRD4, and Bromodomain testis-specific protein (BRDT) have been extensively studied in both physiology and disease as members of the BET family (24–26). Notably, BRD4 (Fig. 1) is involved in epigenetic regulation via binding to acetylated histones on DNA. Upon its interaction with acetylated histones, it makes DNA flexible for transcription through its ionic interactions. Through the recruitment of transcription factors resulting in mediator complexes, phosphorylation of RNA polymerase II, and intrinsic histone acetyltransferase activity, it regulates the expression of therapeutically essential gene regulatory networks (27). In recent years, there has been a surge in the prominence of BRD4 research due to its ability to identify acetylated histones. This protein plays a crucial role in transcription, replication, and DNA repair, hence contributing significantly to developmental processes, tissue growth, and numerous physiological mechanisms. Moreover, BRD4 has been linked to the pathogenesis of several diseases, including cancer, viral infection, inflammation, and neurological disorders (28). Therefore, the investigation of highly effective compounds for the modulation of BRD4 functions has garnered significant attention in recent years (27). The inhibition of protein-protein interactions involving BRD4 and acetyl-lysine has shown significant efficacy in impeding cell proliferation in cancer, suppressing cytokine production during acute inflammation, and other related effects. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed to human clinical trials (29, 30).

Fig. 1: BRD4 structure and its isoforms.

Protein isoforms of human (h) BRD4. Sequence lengths and specific regions are represented. BD, bromodomain; CTM, C-terminal module; ET, extra-terminal domain; GPA: glycine-proline-alanine; NLS, nuclear localization signal; SEED, Ser/Glu/Asp-rich region.

3.1. Role of BRD4 in viral genome replication

BRD4, in collaboration with E2, the major regulatory protein in PVs, participates in multiple steps of the HPV life-cycle, including replication initiation, viral gene transcription, and viral genome segregation and maintenance (Fig. 2). BRD4 phosphorylation regulated by protein phosphatase 2A (PP2A) and casein kinase II (CK2) is essential for E1- and E2-dependent origin replication as well as viral gene transcription. Therefore, inhibitors blocking phospho-BRD4 functions or compounds regulating BRD4 phosphorylation are promising candidates for treating human HPV infections and associated diseases.

Fig 2: BRD4 involves multiple-steps of HPV life-cycle.

The viral early protein 2 (E2), is one of the major regulators of the HPV life-cycle and is involved in the viral genome replication. E2 amino-terminal domain, which contains highly conserved amino acid residues is required for the attachment of HPV genomes to mitotic chromosomes, activating the viral transcription and replication (7). E2 interacts directly with BRD4, which acts as a scaffold protein to recruit a variety of chromatin regulators and transcription factors to control transcription (31, 32). The most well-characterized factors recruited by BRD4 are: the Positive transcription elongation factor b (P-TEFb), transcriptional regulators, such as E2, p53 and NFκB, and general cofactor mediators (33). BRD4 strongly binds to E2 proteins from a diverse group of PVs and stabilizes the binding of BRD4 to interphase chromatin (34). The interaction of BRD4 and E2 appears as distinct dots on mitotic chromosomes (35). BRD4 appears to play a cell type- and context-dependent role in HPV genome replication as reported by overexpression studies of viral proteins. BRD4 is localized in nuclear foci together with E1 and E2 when they are co-expressed. But when each protein is expressed individually or in the absence of both, BRD4 is concentrated as small dots throughout the nucleus. However, BRD4 is relocated around the replication foci as soon as HPV genome amplification starts, which indicates that BRD4 is involved in the transition from initial amplification to maintenance replication (36).

BRD4 tethers the viral genome to the active cellular chromatin of the infected cell, to initiate the viral transcription and promote together with the newly translated E2 an extended c-Fos/AP-1 wound healing response (37, 38). BRD4 recruits E1 and E2 to the HPV genome after chromosome attachment, and the E1 helicase activity excites a damage and repair response. BRD4 is displaced to the periphery of the growing foci and seems to be no longer necessary for continuous replication of the genome when the viral genomes replicate and the foci grow larger (20).

BRD4 is the most extensively studied member of the BET family. It plays an important role in controlling the cell cycle of normal mammalian cells and affecting numerous cellular processes, including cell proliferation, apoptosis, and transcription (39, 40).

During mitosis, the replicated HPV episomes need to partition to the nuclei of daughter cells to ensure persistence in both transformed and infected cells. The viral genome needs to be localization within the nuclear envelope following nuclear reassembly to avoid dilution or degradation in the cytoplasm after cell division. The suggested mechanism for long-term maintenance of viral episomal genomes within replicating cells is via E2 protein (41–44). E2 is the key player in the replication of viral DNA by binding cooperatively with viral E1 protein to the viral origin. In addition to this interaction and the minimal origin of replication, a cis minichromosome maintenance element (MME) encompassing multiple E2 binding sites is also required for long-term maintenance of the viral episomes and to support the transient replication of viral plasmids (45). MME attaches to the mitotic chromosomes in the presence of E2 protein. Moreover, the function of the chromatin attachment associated with the stable episomal is maintenance of the viral plasmids (42). Therefore, it was concluded that E2 facilitates the viral genome segregation by binding the viral genomes to the condensed mitotic chromatin. BRD4 plays a major role as a cellular receptor for viral genome and E2 on mitotic chromosomes. You et al. described that blocking the interaction of E2 to BRD4 inhibited transformation of C127 mouse cells by cloned viral Boven papilloma virus-1 (PV-1) DNA. Moreover, Inhibition of the BRD4/E2 interaction prevented the mitotic chromosomal targeting the viral genome and E2 protein. They also demonstrated that the HPV16 E2 protein is associates with BRD4 (18). The interaction between BRD4 and BPV-1 E2 was confirmed in vitro and in vivo. The E2 binding region of BRD4 was mapped to a 139 aa C-terminal domain. BRD4 binds to the E2 N-terminal transactivation domain, this domain is responsible for the colocalization of E2 with BRD4 on mitotic chromosomes.

Although the colocalization of E2 with BRD4 is mainly occurs in mitotic cells, where both of them are observed as high-density punctate dots on mitotic chromosomes, at what phase of the cell cycle the association of both proteins is conducted remains poorly understood.

Sakakibara et al. showed that the BRD4 protein localizes to nuclear foci recruiting proteins associated with the host DNA damage response. These foci are formed by the expression of the HPV16 E1 and E2 replication proteins in keratinocytes (36). Although BRD4 is crucial for these foci formation, when E1 and E2 start the viral DNA amplification, cellular factors associated with homologous recombination and DNA synthesis are recruited and displace BRD4 from the foci. They also compared these nuclear foci with others in differentiated HPV-infected keratinocytes and showed that there is a switch between early BRD4-dependent foci and mature BRD4-independent replication foci.

In human C-33 cervical cells, Jang et al. used FANCD2 ChIP-chip to map fragile sites that oncogenic viruses target to integrate the genome into a number of large regions of the human genome. These targets frequently contain deletions, are associated with common fragile sites and have increased rates of asynchronous DNA replication (34). They proposed that HPV replication adjacent to highly susceptible fragile sites, increases the chances of integration significantly as is found in HPV-associated cancers.

McKinney and colleagues in 2016 assessed the role of BRD4 in the initial infectivity of keratinocytes using an HPV18 quasivirus-based genome delivery system and showed that BRD4 activates viral transcription and replication independently of the functional interaction between BRD4 and the E2 protein (46). Wu et al. reported that BRD4 uses a phosphorylation-induced switch mechanism to recruit HPV-E2 protein to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Active AP-1 and NF-κB are recruited to their target sequences via BRD4-dependent mechanism, which enhances MMP-9 expression upon keratinocyte differentiation. This process is triggered by the re-localization of NF-κB from the cytoplasm to the nucleus as well as by the replacement of AP-1 family members JunB and JunD by c-Jun. Compounds targeting phospho-BRD4 effectively block E2-regulated viral and cellular gene transcription (37).

Dooley et al., 2016 detected binding of the super-enhancer markers BRD4, H3K27ac and MED1 at the tandemly integrated copies of HPV16 in W12 subclone 20861 cervical neoplasia cells. BRD4 was displaced from the HPV integration site after treating 20861 cells with bromodomain inhibitors, which inhibited also E6/E7 transcription and cellular proliferation. They concluded that viral transcription is activated by BRD4 at this integration site (47).

3.2. Role of BRD4 in viral transcription

HPV genome contains nine to ten open reading frames encodes for early and late genes. The early genes produce the regulatory proteins E1–E8 in the basal cell layer, whereas the late genes take place within the differentiated keratinocytes in the squamous epithelium expressing structural proteins, L1 and L2, which contribute into the viral capsid structure (48). HPVs dependent on the host cell transcription machinery to replicate since their viral capsids do not contain any viral transcription factors (49).

Wu et al. analyzed cellular factors from transcriptionally silenced HPV chromatin, which can trigger gene activation using an in vitro-reconstituted HPV chromatin transcription system, and illustrated that initiating of HPV transcription is driven by the critical cellular factor AP-1 (50). AP-1-dependent HPV chromatin transcription approach was beneficial in the detection of the essential cellular elements for E2-mediated repression of HPV transcription. Correspondingly, BRD4 as an E2 core-pressor was identified by Wu and colleagues and then confirmed by Smith et al., using unbiased genome-wide siRNA knockdown screen (51). Purification and reconstitution of various dimeric AP-1 complexes formed between Fos (FosB, c-Fos, Fra-1, and Fra-2) family proteins and Jun (JunB, c-Jun, and JunD) (52) demonstrated clearly the binding of AP-1 and AP-1-binding sites on HPV genome (53).

While C-terminal motif of BRD4 interacts with the N-terminal domain of all E2 proteins, BRD4 binds to the C-terminal domain of low-risk E2 only when the N-terminal casein kinase II (CK2) phosphorylation site cluster (i.e., N-terminal phosphorylation sites [NPS]) of BRD4 is unphosphorylated as this exposes the internal basic residue-enriched interaction domain (BID) of BRD4 (54). The C-terminal domain of high-risk (HR) E2 binds to phosphorylated as well as unphosphorylated BRD4. Via structure-guided mutagenesis analysis and the sequence alignment between LR-E2 and HR-E2, phospho-NPS-interacting residues were mapped in HPV16 E2 to two basic residues, K306 and K307, and in HPV18 E2 to R307 and K308, which are located on the C-terminal end of the E2 DNA-binding domain (DBD). These residues are conserved among the BID and BRD4 itself, as well as the phospho-NPS-binding domain of the C-terminal region of p53. In addition, they do not interfere with E2 binding to DNA and (37).

HPV11 E2 is converted into a phospho-NPS-interacting protein when the two basic residues N304 and D305 are mutated to KK as appears in HPV16 E2, indicating the crucial role of these residues in building specific contact with phosphorylated residues in BRD4 and E2. The interaction of E2 with phospho switch-regulated BRD4 potentially crucial for HPV life-cycle where the BRD4-E2 complex is involved in host and viral gene transcription (Fig. 2). This interaction is fine-tuned by the opposing activity of PP2A and CK2 during the differentiation of epithelial layer.

Several reports suggested the presence of viral integration sites hotspots in the host genome (55, 56). HPV integration sites found to be FANCD2-associated genomic regions susceptible to enhancer-enriched genomic regions and to tandem repeat formation and (57). This hypothesis was also supported by Doolittle-Hall et al., 2015 who reported the enrichment of HPV integration sites around genes.

4. BRD4 regulates HIV transcription

Following infection, the HIV undergoes integration into the genome of the host cell and then utilizes the host machinery to regulate transcription. The present ARTs are designed to impede the enzymatic activities of the virus, therefore obstructing the reproduction of HIV-1 and arresting a decline of the host’s immune system. Nevertheless, it has been shown that individuals experience viral rebounds when therapy is discontinued, as a result of the reactivation of dormant HIV-1 proviruses. Although ART has proven to be the most effective approach in managing HIV replication, it does not possess the ability to eliminate the virus (i). Furthermore, it does not impact the stable cellular reservoirs where the virus can persist (ii), nor does it demonstrate efficacy in addressing the inflammation caused by HIV or the associated complications arising from the residual reservoirs (iii) (58). Furthermore, the cessation of ART always leads to the resurgence of viral replication.

The expression of the HIV provirus is controlled by many host epigenetic and transcriptional mechanisms. It is noteworthy that the HIV Tat (Trans-Activator of Transcription) protein has the ability to interact and activate the cellular components inside the host. This process leads to an upregulation in the transcription of HIV genes, ultimately culminating in the generation of new viruses. The use of a chemical inhibitor, namely Didehydro-cortistatin A, has been shown to effectively impede the activity of Tat, a crucial viral protein involved in the transcription of HIV. This inhibition leads to the suppression of Tat’s ability to activate transcription and subsequently induces a state of epigenetic repression in the HIV promoter. It is worth noting that several of diminutive antisense RNAs have been discovered, which have an affinity for the HIV promoter, hence facilitating the inhibition and/or repression of HIV transcription (58). In contrast, only a limited number of techniques have been developed to target the epigenetic control of HIV with the aim of inducing a more profound state of latency.

BRD4 has been implicated in HIV transcriptional control in a number of studies. Competing with Tat for cellular P-TEFb/CDK9, BRD4 was demonstrated to inhibit HIV transcription and promote latency. Brass et al., found that BRD4 depletion enhances HIV-1 infection using either of two orthologous large-scale siRNA libraries (59). Furthermore, BRD4 was identified as a negative regulator of HIV-1 replication (60). Inhibition of BRD4 via JQ1, a pan-BET inhibitor, or RNA interference increased proviral transcriptional elongation and alleviated HIV-1 latency in cell line models. Combination of JQ1 with the NF-κB activators, phytohemagglutinin or Prostratin, enhanced the reactivation of latent HIV-1 in primary human T cells (60). Another study supported the role of BRD4 inhibitor, JQ1, in activation of HIV latency through antagonizing BRD4 inhibition of Tat-transactivation (61). The short isoform of BRD4 was shown to be a corepressor of HIV transcription during latency by tethering SWI/SNF to latent HIV chromatin via binding to the Brahma Related Gene 1 ATPase. Therefore, inhibition of BRD4 disrupts the SWI/SNF nucleosome remodelers at the latent HIV promoter (62). Bisgrove et al., 2007 illustrated that HIV-1 transcription elongation can be suppressed by BRD4, specifically via competition with Tat for binding to cyclin-dependent kinase 9 (CDK9)/p-TERb. Highlighting the involvement of this epigenetic reader in the regulation of HIV-1 transcription (63). The pan-BET inhibitor JQ1 modulates BRD4, which was at least partially mediated by reducing the competition between BRD4 and Tat. In addition, Tat-independent mechanism for JQ1-induced HIV reactivation is also reported (64).

Our team, like others, has used computer-aided design to develop BRD4-targeting small molecule inhibitors. We have reported a new class of small-molecule modulators that more selectively target BRD4 with the lead compound named ZL0580. Notably, in contrast to JQ1, the molecule interacts with BRD4 in a different manner that improves HIV transcriptional suppression. The mechanisms of action of ZL0580 were partially elucidated in our published studies (65, 66). A proposed model for the mechanisms of ZL0580 is summarized in Fig. 3. In contrast to JQ1, a broad-spectrum BET inhibitor that targets both BD1 and BD2 of all four BET proteins, ZL0580 exhibits selective binding to BD1 of BRD4 without significant affinity towards other BET proteins. Using the determined BRD4/BD1 co-crystal structure (67), our docking analysis indicated that ZL0580 binds to a new non-acetyl-lysine (KAc) binding site of BRD4 BD1 that is distinct from the classic KAc binding site recognized by JQ1 (58, 65). This new binding site critical for protein-protein interaction modulation was further confirmed with the co-crystal structure of its close analog ZL0590 in complex with human BRD4 BD1 (68) (Ref). The transcriptional activity of ZL0580 has been shown to be reliant on BRD4. This has been demonstrated in our studies, including the deletion and overexpression of BRD4, which have verified the suppressive impact of ZL0580 on HIV. Furthermore, the inhibitory efficacy of ZL0580 was validated against both transcriptionally active and latent HIV in multiple cell models, such as J-Lat cells, human peripheral blood mononuclear cells/primary CD4⁺ T, and human myeloid cells/microglia (58, 65, 66).

Fig 3: Proposed mechanisms of ZL0580 in suppressing HIV.

ZL0580 exhibits binding affinity towards BRD4, specifically targeting a location of the non-KAc site other than the classic KAc binding site on BRD4. This interaction results in the augmentation of binding between ZL0580 and its associated binding partners (P-TEFb/CDK9) for protein-protein interactions. Nevertheless, it inhibits the interaction between Tat and BRD4, leading to the downregulation of the ELL2 protein via a decrease in protein stability. The combination of diminished Tat-CDK9 interaction and decreased ELL2 expression results in a decrease in RNA polymerase phosphorylation at the HIV long terminal repeat (LTR) and a disruption in the elongation of HIV transcription.

Importantly, we examined HIV-suppressing activity of ZL0580 in combination with ART on ex vivo CD4⁺ T cells isolated from three ART suppressed HIV-infected participants. We showed that combination treatment with ZL0580 and ART accelerated HIV-1 suppression and delayed virus rebound by 8, 9, and 15 days, respectively, compared to ART alone (65).

5. HPV integration and MYC

MYC protooncogene is involved in the regulation of a variety of cellular processes, including metabolism, differentiation, apoptosis, and proliferation (69). Cells function normally with homeostatic levels of MYC. However, MYC overexpression may enhance tumorigenesis. MYC stability is regulated through phosphorylation: functional activation and stabilization are controlled by phosphorylation at Ser62, while Thr58 phosphorylation signals degradation (70, 71).

MYC and BRD4 are involved in a wide variety of diseases, especially cancer. As transcriptional regulatory factors controlling numerous cellular functions, they play critical roles in the posttranslational and chromatin regulation of many other proteins involved in various biological processes. BRD4 directly regulates transcription by phosphorylation of many other interacting proteins via its kinase activity, such as transcription elongation factor P-TEFb, transcription factors MYC and TAF7, and the RNAPII CTD (72). Furthermore, wang et al., 2022 used whole-genome sequencing, whole-transcriptome sequencing, high-throughput HPV captured sequencing and OncoScan microarrays to report human papillomavirus features and genomic landscape in small cell cervical carcinoma (SCCC). They illustrated that 37.9% of SCCC samples had a much greater percentage of HPV-integrated breakpoints situated in MYC family genes (73).

6. BRD4 regulates MYC activity

MYC and BRD4 mutually regulate each other and have overlapping effects. MYC regulates BRD4 function by inhibiting its histone acetyltransferase (HAT) activity. BRD4 helps maintain homeostatic levels of MYC by influencing MYC stability and activating MYC transcription.

The enzymatic activities of BRD4 and its structure as bromodomains with intrinsically disordered regions, in addition to its ability to interact with key transcription factors, chromatin remodelers/modifiers and coactivators contribute to its pleiotropic effects on stability, transcription and regulation of MYC.

BRD4 recruits P-TEFb to mitotic chromosomes through interactions with cyclin CDK9 and T1 resulting in increased expression of growth-promoting genes (74). Chromosomal rearrangements of BRD4 to the nuclear protein in the testis (NUT) locus on chromosome 15q14 generates a BRD4-NUT fusion protein in approximately 80% of cases that results in NUT midline carcinoma (NMC) and c-MYC overexpression (75). Inhibition of BRD4 downregulates MYC transcription and subsequent MYC-dependent target genes (76). This holds great promise for cancer treatment considering the widespread pathogenicity of MYC in cancers.

As a protein with numerous functions, BRD4 is a BET protein. It has also been described as a mitotic bookmark, a DNA damage response factor, a recruitment and scaffolding factor, and a transcription cofactor. As befits its multiple roles, transcription, DNA replication, cell cycle, and signaling during stress reactions are just a few of the several biological processes in which BRD4 is active. BRD4 malfunction has therefore been connected to a wide range of illnesses, including different malignancies, heart, kidney, lung, and inflammatory diseases, auto immunological diseases, and neuro-degenerative disorders (28, 77). Initially, BRD4 was described as a scaffolding factor that attracted several important chromatin and transcriptional regulatory components. However, it was identified that BRD4 contains two intrinsic enzymatic activities, kinase and HAT activity. This property enables it to dynamically control its interacting partners. The enzymatic properties of BRD4 distinguish it from other BET proteins. BRD4 directly controls transcription by phosphorylating many of its interaction partners, including the transcription factors MYC and TAF7, the transcription elongation factor p-TEFb, and most significantly the RNAPII CTD (72). BRD4 controls chromatin architecture and acetylates nucleosomes through its HAT activity (78). Thus, BRD4 plays crucial functions in directly controlling chromatin structure and the transcription machinery through its distinctive binding domains and varied enzymatic activity. BRD4 controls the MYC directly and plays a crucial regulatory role at across the genome, in addition to controlling transcription at promoters (76, 79, 80). The discovery that BRD4 could modulate MYC transcript levels sparked intense interest in BRD4 as a pathway to control MYC, an important oncogene that is notoriously difficult to control. Small-molecule BET inhibitors like JQ1 and I-BET, which imitate the acetyl moiety, bind to BET proteins to occlude their acetyl lysine-binding pocket, and potently restrict the proliferation of malignant cells by lowering the production of oncogenes like MYC, made it easier for BRD4 to regulate MYC levels (81). In MYC-driven malignancies, BRD4 is therefore being researched as a potential therapeutic target. It was found that BRD4 maintains homeostatic levels of MYC protein by directly destabilizing it by phosphorylating it at a location that leads to ubiquitination and destruction (82).

MYC transcription is regulated by BRD4 through nucleosome clearance at the MYC locus via phosphorylation of RNAPII CTD at Ser2 and by its HAT activity which leads to topoisomerase activation. BRD4 also regulates Pol II pause release by recruitment of JMJD6 or P-TEFb (83). Moreover, BRD4 regulates MYC superenhancers (SE) activity, and is involved in the recruitment of both P-TEFb and Mediator to SEs. Additionally, BRD4 incorporation into SE condensates can be increased by phosphorylation of RNAPII CTD, which possibly enhances MYC transcription (84). Furthermore, BRD4’s could inhibit methylation of important regulatory regions in SEs by recruitment of Glutamine and Serine Rich 1 (QSER1) protein resulting in elevated MYC expression (85). BRD4 could elevate MYC expression by the formation of extrachromosomal circular DNA (ecDNA) hubs which are known to drive cancer progression (86, 87).

MYC can regulate transcription either directly by interacting with critical transcription regulators, including BRD4, P-TEFb, RNAPII and Mediator or by affecting chromatin remodelling through recruiting chromatin re-modelling machines (CRMs) and HATs (82). It is involved in pause release through P-TEFb and plays a significant role in enhancing transcription through its activation of topoisomerases (Top I & II) (88).

MYC also increases mRNA CAP methylation of P-TEFb and TFIIF enhancing their translation. MYC levels could be enhanced through N6-methyladenosine (m6A) RNA writer–reader machinery by isolating m6A-MYC RNA in condensates and preventing their degradation as well as by increasing MYC transcription (89). Therefore, BRD4 and MYC regulate cellular programs through their interactions with multiple influencers and modifiers of transcription and translation leading to elevated MYC expression and oncogenesis (90–92).

BRD4 regulates MYC and its target genes by phosphorylating MYC at Thr58, resulting in MYC ubiquitination and degradation. Notably, MYC protein levels increase by BRD4 degradation, but not inhibition. Reciprocally, MYC inhibits BRD4’s HAT activity, indicating that MYC transcription is regulated by limiting chromatin remodeling associated to BRD4 on its locus (82). However, these changes may be cell type specific since several reports indicated that BRD4 PROTACs treatment reduced MYC protein expression (93, 94).

The various domains of both BRD4 and MYC and post-translational modifications in these domains play a major role in the ultimate functions of the two proteins. Understanding the wide variety of BRD4 and MYC functions, the complex interaction between them, the interwoven, multifaceted, regulatory pathways between them hold great promise toward the therapeutic application of these proteins to achieve clinical success, particularly dysregulation of these proteins have been linked to numerous diseases.

7. Inhibition of BRD4

Over the past decade, targeting BRD4 has attracted significant interest in both academic and pharmaceutical settings. The number of BET inhibitor molecules has expanded dramatically and represents a promising therapeutic target for various diseases as well as exploring the biological functions of BET. Promising efforts have been devoted to producing BRD4 inhibitors and many candidates have progressed to human clinical trials (27).

Most BRD4 inhibitors simulate and compete with acetyl-lysine to bind to BRD4 and then block the interactions between acetyl-lysine and BRD4. As the O atom of the acetyl group does, BRD4 inhibitors form critical hydrogen bonds with Asn140 and Tyr 97 through a unique head moiety (95).

Rataj et al., 2018 studied the inhibition of BRD4 by JQ1 to modulate E6 transcription and demonstrated a strong suppressive effect on E6 expression in CC10/CDDP-R and SiHa cell lines. Inhibition of E6 expression by JQ1 treatment enhanced cisplatin chemoresponse and reversed the drug resistant phenotype in only HPV positive cisplatin-resistant variants C33A cervical cancer cells. Therefore, they concluded that inhibition of BRD4 could represent a potential therapeutic option by enhancing the treatment response of cisplatin in cervical cancer cells (96).

Latif et al., 2021 demonstrated that combination therapy of MDM2 inhibitors (Nutlin-3) activating p53 and BET inhibiter targeting BRD4 presents enhanced anti-acute myeloid leukemia activity, resulting from (CPI203) ability to release BRD4-mediated repression of p53 target genes and enhance p53-induced apoptosis. Therefore, BRD4 inhibiter and wild-type TP53 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML (97). A similar study used the BRD4 inhibitor ZEN-3365 (98).

Inhibition of BRD4 and acetyl-lysine interaction blocked cytokine production in acute inflammation and reduced cell proliferation in cancer (27).

BRD4 inhibitors and vorinostat inhibited HPV genome amplification in naturally HPV infected cells. Recently, we assessed whether these naturally HPV infected cells can serve as an in vitro pathological cell model for screening anti-HPV drugs. Since viral genome amplification is required for the HPV life cycle and is also easy to quantify with qPCR, we would like to test the possibility of viral genome amplification as a measurement or readout for anti-HPV screening. Since BRD4 is involved in multi-steps of HPV life cycle (54), we tested our new BRD4 inhibitors (ZL0580), JQ1, and vorinostat in patient-derived cells. In the 2D cultures, our results showed that these drugs had no effect on HPV18 DNA copies compared to DMSO control (Fig. 4). Interestingly, these drugs were able to significantly decrease viral genome copy compared to control in ALI 3D cultures (Fig. 4). Thus, our results demonstrated that naturally HPV infected cells can be used for anti-HPV testing when the cells are cultured under ALI 3D where HPV life cycle occurs. More specifically, ZL0580 exhibited similar suppressive effect on HPV replication under 3D cultures, not in 2D cultures (Fig. 4).

Fig. 4. BRD4 inhibitors inhibit HPV DNA amplification in patient-derived and naturally HPV infected cells under 3D cultures, not 2D cultures.

No inhibition effect of JQ1, ZL0580 and vorinostat on HPV DNA in 2D cultures of naturally HPV-18 infected, patient-derived cells (left). Cells were treated by DMSO, JQ1, ZL0580 or vorinostat (5 uM) for 24 hrs. HPV-18 DNA copies were determined by qPCR. Inhibition of HPV-18 DNA copies by JQ1, ZL0580 and vorinostat in ALI 3D cultures. ALI 3D cultures of patient-derived cells were exposed to DMSO or ZL0580. Drug treatment was added at day 11 to 18 of ALI 3D cultures. Cellular DNA was collected at day 18. HPV18 DNA copies were determined by qPCR using a HPV18 plasmid DNA as a control.

An approach utilizing cell conditional reprogramming (CR) coupled with the air-liquid interface (ALI) might provide an efficient human physiological cell model (99) to examine the effect of BRD4 inhibitors on both HPV and HIV viral life cycle and their role in cancer initiation (Fig. 4 and 5). As we discussed above, JQ1 activated HIV transcription and inhibited HPV DNA amplification, while our new selective BD1 modulator, ZL0580, inhibited HIV and HPV. Thus, our proposed cell model in Fig. 5 would be unique for drug discovery of dual inhibitors of HIV and HPV that are unmet needed for treating HPV associated cancer in PLWH.

Fig. 5.

Experimental systems for studies of how HIV affects HPV life cycle and oncogenesis and discovery of dual inhibition of HIV/HPV. HPV positive CR cells from patients can be used to establish and 3D based culture systems. HPV cells will be cocultured with HPV in the presence of HIV isolates and mutants. HIV factors (proteins or RNAs) including gp120, Tat, Nef, TAR, Vpr can be used in these systems for studies of regulation and inhibition of HPV viral gene expression, replication and biological changes of host cells with naturally HPV infected cells after coculture.

CONCLUSION

The BRD4 protein is critically involved in multiple processes in the HPV infectious cycle including transcription, replication and genome partitioning. Comprehension of how BRD4 functions are utilized to facilitate the differentiation-dependent viral lifecycle has the potential to provide insights into the cellular functions of this pivotal epigenetic regulator. Further extensive investigation of the BRD4 cellular functions and action mechanisms of novel inhibitors could lead to the development of anti-cancer/anti-viral therapies using patient-derived naturally HPV infected cells with cultures with HIV isolates or HIV factors. These would lead to drug discovery of dual inhibition of HIV and HPV for treating HPV infection and HPV associated cancer in PLWH. Recent studies demonstrated that HPV integrates predominantly into SOX2, TP63, MYC, and GFGR3 loci in HPV positive oral cancers, thus selective BRD4 targeting may benefit advanced HNSCC with integrated HPV genomes.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.