Abstract 203: Synergism Of TNFα And IFNγ Elicits Transcriptional Cooperativity At The CXCL9,-10,-11 Locus In Human Endothelial Cells And Is Ultrasensitive To BRD4 And CBP/P300 Inhibition.

Abstract

Background/Rationale: Combinatorial signaling by proinflammatory cytokines drives the pathogenesis of many cardiovascular diseases ranging from acute SARS-COV-2 vascular complications to chronic atherosclerosis. TNFα and IFNγ synergistically induce genes that amplify inflammatory cascades, causing tissue damage and high mortality in humans. Characterization of transcriptional mechanisms governing synergistic gene induction holds the promise of revealing novel therapeutic targets while also improving our understanding of how inflammatory states are propagated. Objective: To delineate unique mechanisms of synergistic gene transcription in human aortic endothelial cells (HAECs) exposed to high levels of both TNFα and IFNγ. Methods & Result: Using RNA-sequencing, we defined the dynamic transcriptome in HAECs stimulated with TNFα, IFNγ, or combination for one hour. We devised a novel statistical approach that generates a positive cooperativity score (PCS) for each gene in order to define the TNFα/IFNγ synergy signature in HAECs. This approach revealed the chemokines CXCL9, -10, -11 as highly synergistic in their expression (PCS=61.9, 18.3, 8.9; log 2 [Combination/No Treatment]=8.1, 2.1, 3.1 respectively), in contrast to SELE , a TNFα-induced gene (PCS=0.4; log 2 [Combination/No Treatment]=6.6). Furthermore, pathway analysis revealed strong enrichment for chemotaxis programs among the synergy gene set. To deconvolute transcriptional coactivator dependencies at these exemplary genes, we conducted a targeted mini-screen of transcriptional inhibitors along with biochemical modelling of inhibitor dose-response curves. We discover that CXCL9, -10, -11 induction is sensitively disrupted by inhibition of BRD4 and CBP/p300 (hill coefficient (HC) > 2) coactivators as compared to SELE (HC<2). Conclusions: These results provide definitive evidence of acute transcriptional synergy at the chemokine genes CXCL9, -10, -11 elicited by TNFα/IFNγ activity in HAECs. As these chemokines are associated with poor outcomes in sepsis, these results also implicate targeted inhibition of transcriptional coactivators as a potential therapeutic strategy for inflammatory cardiovascular diseases.