Background/Purpose: Proteins motifs bromodomain 1 (BD1) and BD2 within the BET family proteins regulate gene expression of inflammatory cytokines and lymphocyte proliferation by binding to acetylated histones and transcription factors. Recent reports on BD1/BD2 inhibitors suggest their potential as therapeutic drugs for the treatment of blood cancer and inflammatory diseases. Although existing compounds with increased selectivity for BD2 showed better safety profiles than BD1/BD2 dual inhibitors such as JQ-1, still little is known about the specific function of BD2 due to incomplete selectivity for BD2 over BD1 with existing compounds. We generated a compound that shows the highest selectivity for BD2 compared to ones previously reported. We evaluated our novel BD2 inhibitor on lymphocyte in vitro, and on in vivo mouse models aiming to understand specific contribution of BD2 in chronic inflammatory diseases.

Methods: Human peripheral blood mononuclear cells (PBMCs) from healthy donors, patients with IgG4-related disease (IgG4-RD), or rheumatoid arthritis (RA) diagnosed at Keio University Hospital were isolated to B cells or CD4 T cells. Effects of our novel BD2 inhibitor (BD2i) on differentiation of plasma cells and Tfh/Tph cells were analyzed in vitro. Furthermore, in vivo efficacies of BD2i were evaluated in nitrophenyl ovalbumin (NP-OVA) immunization-induced antibody production model in C57BL/6 mice and Lupus-like nephritis model in NZB/WF1 mice.
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Results: Our BD2i demonstrated high selectivity for BET-BD2 and BET-BD1 in cell-free binding assays (IC₅₀ = 1.4 nM for BD2, IC₅₀ > 10 µM for BD1). In human primary cell-based assay, the BD2i inhibited differentiation of plasma cells (IC₅₀ = 33 nM) and IL-21⁺ Tfh/Tph cells (IC₅₀ = 5/11 nM, respectively) with comparable IC₅₀ values as those of BD1/BD2 dual inhibitors. The BD2i inhibited in vitro differentiation of plasma cells and Tfh cells from patients with IgG4-RD and RA, as well as those from healthy donors. Notably, the BD2i showed little impact on the proliferation of B cells and CD4 T cells while BD1/BD2 dual inhibitors strongly suppressed cell proliferation. Moreover, the BD2i inhibited production of antigen-specific antibodies in NP-OVA-immunized mice at oral doses of 1 mg/kg (q.d.) or higher, and significantly suppressed nephritis in NZB/WF1 mice at oral doses of 3 mg/kg (q.d.) or higher, correlated to the reduction in the frequency of helper T cells and plasma cells. No adverse findings were observed in NZB/WF1 mice following oral administration of BD2i at 30 mg/kg (q.d.) for 18 weeks.

Conclusion: Our findings suggest that BD2 of BET family proteins plays a crucial role in the pathogenesis of chronic rheumatic diseases such as IgG4-RD, RA, and Lupus nephritis by specifically regulating pathogenic cell differentiation rather than cell proliferation. Therefore, selective BD2 inhibitors that suppress differentiation of pathogenic effector lymphocytes, including plasma cells and Tfh/Tph cells, would be attractive new therapeutics for the treatment of rheumatoid diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bromodomain-2-of-bet-proteins-plays-a-crucial-role-in-follicular-peripheral-helper-t-cell-and-plasma-cell-differentiation/