Targeting epigenetic regulation via Bromodomain and Extraterminal (BET) domain inhibition for treatment of GVHD

摘要 急性移植物抗宿主病（aGVHD）是由于供体T细胞对宿主组织的同种异体反应性而发生的， 异基因干细胞移植（alloSCT）后非复发死亡的主要原因。约 50%的患者对一线皮质类固醇治疗无反应，被认为是"类固醇难治性"， 局限于没有有效的标准疗法。我们的长期研究目标是识别和评估创新 通过预防和/或消除alloSCT后aGVHD毒性来改善患者结果的方法。的 拟议的研究提出了开创性的临床和翻译方法，以靶向表观遗传调控， 使用新的非免疫调节剂通过溴结构域和末端外（BET）结构域抑制炎症介质 苯二氮卓类结构化PLX（51107和2853）预防和减轻aGVHD。PLX拥有改进的 药物代谢动力学和耐受性特征。我们观察到，BET PLX抑制导致T细胞增殖和促炎细胞因子分泌的有效抑制 IFN-g、IL-6和TNF-α的水平，而不影响T细胞活力。我们的数据还表明，BET抑制与 PLX显著下调T细胞共刺激基因、主要炎性细胞因子的转录， 细胞周期调节器。重要的是，我们发现BET抑制降低了T细胞增殖并抑制了T细胞增殖。 炎症不依赖于STAT-1。因此，我们针对JAK/STAT阻断与最近FDA批准的 JAK1/2抑制剂鲁索利替尼和观察到的双重BET和JAK1/2抑制对T细胞的协同作用 增殖我们假设用PLX抑制BET是一种可行的、有效的策略，以减轻T细胞 介导的aGVHD炎症。此外，我们提出了临床前分析，以评估 双重BET/JAK1/2抑制的协同机制和耐受性。拟议研究测试的目标1 我们在1b/2期原则性临床试验中对类固醇难治性aGVHD患者的假设， 单剂PLX51107。相关研究旨在评估1）对治疗的反应和2）免疫 T细胞亚群的重建。在目标2中，我们将使用aGVHD的临床前模型来检验以下假设： BET抑制通过下调Th1和Th17致病性T细胞导致aGVHD存活的改善 同时维持Treg介导的耐受性。我们观察到， Th1/Th17促炎基因如IFN-γ、IL-17和IL-2以及共刺激分子CD40 L 用PLX处理的T细胞在体外。我们建议分析PLX对单个T细胞亚群及其 在体内aGVHD发病机制中的意义。目标3将1）检验双重BET和JAK 1/2 抑制将减少GVHD并延长治疗反应的持续时间;和2）评估 联合治疗对Treg和效应T细胞功能的影响。这些实验将决定未来 T细胞亚群定向治疗aGVHD的适用性以及为未来临床的发展提供信息 联合BET和JAK 1/2抑制用于aGVHD预防或治疗适应症的试验。

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 determine the future applicability of T cell subset directed therapy for aGVHD as well as inform the development of future clinical trials combining BET and JAK1/2 inhibition for aGVHD prevention or treatment indications.