Abstract Acute Graft-versus-Host Disease (aGVHD) occurs due to donor T cell alloreactivity against host tissues and is the major cause of non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Approximately 50% of patients are not responsive to front-line corticosteroid therapy and deemed “steroid-refractory” with limited to no effective standard therapies. Our long-term research goal is to identify and evaluate innovative approaches to improve patient outcomes by preventing and/or abrogating aGVHD toxicity post-alloSCT. The proposed research presents pioneering clinical and translational approaches to target epigenetic regulation of inflammatory mediators via Bromodomain and Extraterminal (BET) domain inhibition using novel non- benzodiazepine structured PLX (51107 and 2853) to prevent and mitigate aGVHD. PLX boasts improved pharmacokinetic and tolerability profiles to benzodiazepine-scaffolded BET inhibitors. We observed that BET inhibition with PLX results in potent suppression of T cell proliferation and pro-inflammatory cytokine secretion of IFN-g, IL-6, and TNF-α without affecting T cell viability. Our data also demonstrate that BET inhibition with PLX significantly downregulates transcription of T cell costimulatory genes, major inflammatory cytokines, and cell-cycle regulators. Importantly, we identified that BET inhibition decreases T cell proliferation and dampens inflammation independent of STAT-1. Thus, we targeted JAK/STAT blockade with the recently FDA approved JAK1/2 inhibitor ruxolitinib and observed synergistic effects of dual BET and JAK1/2 inhibition on T cell proliferation. We hypothesize that BET inhibition with PLX is a feasible, effective strategy to mitigate T cell mediated aGVHD inflammation as a single agent. Further, we propose preclinical analyses to assess the synergistic mechanisms and tolerability of dual BET/JAK1/2 inhibition. Aim 1 of the proposed research tests our hypothesis in a Phase 1b/2 proof-of-principle clinical trial for patients with steroid-refractory aGVHD with single agent PLX51107. Correlative studies are designed to assess 1) response to therapy and 2) immune reconstitution of T cell subsets. In Aim 2, we will use preclinical models of aGVHD to test the hypothesis that BET inhibition results in improvement in aGVHD survival by downregulating Th1 and Th17 pathogenic T cell responses while maintaining Treg mediated tolerance. We observed a very strong reduction in expression of Th1/Th17 pro-inflammatory genes such as IFN- g, IL-17 and IL-2 as well as co-stimulatory molecule CD40L with PLX treated T cells in vitro. We propose to analyze the effects of PLX on individual T cell subsets and their implications in aGVHD pathogenesis in vivo. Aim 3 will 1) test the hypothesis that dual BET and JAK1/2 inhibition will reduce GVHD and prolong duration of treatment response; and 2) assess the effects of combination therapy on Treg and effector T cell function. These experiments will determin...