The suppression of Brd4 inhibits peripheral plasma cell differentiation and exhibits therapeutic potential for systemic lupus erythematosus

Background and purpose: To investigate the role of bromodomain-containing protein 4 (Brd4) in regulating B cell differentiation and its therapeutic potential for B cell-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Experimental Approach: Human and murine B cells were purified and cultured with different stimuli. B cell surface markers, proliferation and apoptosis were estimated by flow cytometry. Gene expression was measured by quantitative real-time PCR. Brd4 binding sites were analysed by the luciferase reporter assay and the chromatin immunoprecipitation (ChIP) assay. PFI-1 or JQ1 was used to inhibit Brd4. Mice with B cell-specific deletion of the Brd4 gene (Brd4flox/floxCD19-Cre+/-) and MRL/lpr mice were used to perform the in vivo experiments. Key Results: Brd4 inhibition suppressed plasmablast-mediated plasma cell differentiation but did not influence proliferation or apoptosis in healthy human and murine CD19+ B cells. PFI-1 treatment reduced the secretion of IgG and IgM in the supernatants of costimulation-induced B cells. Mechanistically, Brd4 regulates the terminal differentiation of B cells into plasma cells by targeting BLIMP1 by directly binding and activating the endogenous BLIMP1 promoter. Interestingly, PFI-1 treatment decreased the percentages of plasmablasts and plasma cells from patients with SLE. PFI-1 administration reduced the percentages of plasma cells, hypergammaglobulinemia and attenuated nephritis in MRL/lpr mice. Pristane-injected Brd4flox/floxCD19-Cre+/- mice exhibited improved nephritis and reduced percentages of plasma cells. Conclusions and Implications: Brd4 is an essential factor in regulating plasma cell differentiation. Brd4 inhibition may be a potential new strategy for the treatment of B cell-associated autoimmune disorders, including SLE.

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.

BRD4 inhibition alleviates mechanical stress-induced TMJ OA-like pathological changes and attenuates TREM1-mediated inflammatory response

The aim of this paper was to investigate the protective effects of bromodomain containing 4 (BRD4) inhibition on the temporomandibular joint osteoarthritis (TMJ OA) induced by compressive mechanical stress and to explore the underlying mechanism. In vivo, a rat model of TMJ compressive loading device was used and BRD4 inhibitor was injected into the TMJ region. HE staining and micro-CT analysis were used for histological and radiographic assessment. Immunohistochemistry and qPCR were performed to detect inflammatory cytokines expressions. High-throughput ChIP-sequencing screening was performed to compare the BRD4 and H3K27ac binding patterns between condylar cartilage from control and mechanical force groups. In vitro, the mandibular condylar chondrocytes were treated with IL-1β. Small Interference RNA (siRNA) infection was used to silencing BRD4 or TREM1. qPCR was performed to detect inflammatory cytokines expressions. Our study showed that BRD4 inhibition can alleviate the thinning of condylar cartilage and subchondral bone resorption, as well as decrease the inflammatory factors expression both in vivo and in vitro. ChIP-seq analysis showed that BRD4 was more enriched in the promoter region of genes related to the stress and inflammatory pathways under mechanical stress in vivo. Trem1, a pro-inflammatory gene, was screened out from the overlapped BRD4 and H3K27ac increased binding sites, and Trem1 mRNA was found to be regulated by BRD4 inhibition both in vivo and in vitro. TREM1 inhibition reduced the expression of inflammatory factors induced by IL-1β in vitro. In summary, we concluded that BRD4 inhibition can protect TMJ OA-like pathological changes induced by mechanical stress and attenuate TREM1-mediated inflammatory response.

Bromodomain 4 inhibition leads to MYCN downregulation in Wilms’ tumor cells

Wilms’ tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms’ tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, while those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes. MYCN dysregulation has been associated with a number of pediatric cancers including the anaplastic subtype of Wilms’ tumor. In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms’ tumor. Genomic analysis and in vitro experimentation demonstrate that Wilms’ tumor cell growth can be reduced by modulating MYCN overexpression via BRD4 inhibition. We observed a time dependent reduction of MYCN and MYC protein levels upon BRD4 inhibition in Wilms’ tumor cell lines which led to increased cell death and suppressed proliferation. We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels and should be further explored for its therapeutic potential against Wilms’ tumor.