Bromodomain and Extra-Terminal Domain (BET) As a Therapeutic Target in a Mouse Model of Secondary Hemophagocytic Lymphohistiocytosis (HLH)

Secondary hemophagocytic lymphohistiocytosis (HLH) is life-threatening hyperinflammatory syndrome. It is characterized by the ineffective cytotoxic lymphocyte response, leading to sustained IFN-γ secretion and myeloid effector cell activation, causing tissue damage. BET proteins bind acetylated histones and regulate gene transcription. BET inhibitor OPN-51107 selectively blocks BRD4 activity. We aim to evaluate BET inhibition in a murine model of secondary HLH. The disease is induced in C57BL/6 mice by repeated CpG 1826 and anti-IL-10 receptor antibody injections. Mice were randomized into three groups: healthy control, HLH treated with vehicle, or HLH treated with OPN-51107 (10 mg/kg/day by oral gavage starting on day 4). On day 9, mice were sacrificed. Statistical analysis used one-way ANOVA. Single- cell RNA sequencing at different timepoints demonstrated that BRD4 expression increases overtime in different immune cells (Figure 1). BRD4 upregulation is confirmed by flow-cytometry on day 9 after disease-induction. H3K27Ac was measured by flow-cytometry, we observed a significant increase in the marker levels across both adaptive and innate immune cells in HLH vehicle-treated mice compared to healthy control. Vehicle-treated mice developed hepatomegaly (p <0.0001) and splenomegaly (p<0.0001) compared to healthy controls, while OPN-51107-treated mice showed a significant reduction in both liver and spleen enlargement compared to vehicle-treated (liver p=0.0009, spleen p<0.0001). Anemia is a hallmark of HLH. Red blood cell count was significantly reduced in vehicle-treated HLH mice compared to healthy controls (p 0.0001) and BET inhibitor treated mice (p=0.0035). Plasma IFN-γ, IL-6 , and IL-18 and IL-6 were elevated in vehicle-treated HLH mice (IFN-γ p=0.0003; IL-6 p<0.0001; IL-18 p<0.0001) but returned to baseline or were significantly lowered in BET inhibitor–treated HLH mice (IFN-γ p= .0156; IL-6 p=0.0002; IL-18 p=0.0375) (Figure 2). HLH caused expansion of splenic myeloid cells (CD4-/CD8-/CD11b⁺, p=0.0013) and increased their activation status (CD11b+/CD44+, p<0.0001) compared to healthy control. BRD4 inhibition decreased the percentage of positive myeloid cells and their activation status (CD4-/CD8-/CD11b⁺, p=0.0024; CD11b+/CD44+ p=0.0041). Depletion of CD4⁺ T cells (p<0.0001) in vehicle-treated mice versus healthy controls is ameliorated following treatment (p=0.0140). Intracellular IFN-γ in CD8+T cell and TNF-α in myeloid cell was markedly increased in vehicle-treated HLH mice compared to both healthy controls (CD8+/IFN- γ+ p<0.0001; CD4-/CD8-/CD11b+/TNF-α+ p<0.0001) and OPN-51107–treated HLH mice (CD8+/IFN- γ+ p = 0.0003; CD4-/CD8-/CD11b+/TNF-α+ p=0.0002). We demonstrated that BRD4 is overexpressed in different immune cell subtypes and that its inhibition counteracts the clinical hallmarks of secondary HLH in a mouse model.