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)是由于供者T细胞对宿主组织的同种异体反应引起的 异基因干细胞移植后无复发死亡的主要原因。大致 50%的患者对一线皮质类固醇治疗无效，并被认为对类固醇无效 仅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/或消除异基因干细胞移植后移植物抗宿主病毒性来改善患者预后的方法。这个 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 通过溴域和端外(BET)结构域抑制炎症介质的新型非 苯二氮卓构造PLX(51107和2853)以预防和减轻移植物抗宿主病。PLX吹嘘改进了 苯二氮卓类支架BET抑制剂的药代动力学和耐受性。我们观察到，BET PLX抑制可有效抑制T细胞增殖和促炎细胞因子分泌 干扰素-g、IL-6和肿瘤坏死因子-α在不影响T细胞活性的情况下。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、主要炎症细胞因子和 细胞周期调节器。重要的是，我们发现抑制BET会减少T细胞的增殖并抑制 炎症不依赖STAT-1。因此，我们以最近FDA批准的JAK/STAT封锁为目标 JAK1/2抑制剂ruxolitinib及双重BET和JAK1/2抑制对T细胞的协同作用 扩散。我们推测，用PLX抑制BET是一种可行的、有效的策略来减轻T细胞 作为单一介质介导aGVHD炎症。此外，我们建议进行临床前分析，以评估 BET/JAK1/2双重抑制的协同机制及耐受性。拟议研究测试的目标1 我们在1b/2期原则验证临床试验中对激素难治性aGVHD患者的假设 单一代理PLX51107。相关研究旨在评估1)治疗反应和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T细胞改善aGVHD存活率 在保持Treg介导的耐受性的同时做出反应。我们观察到了非常强烈的减少表达 Th1/Th17促炎基因干扰素-g、白介素17和白介素2以及共刺激分子CD40L 用PLX处理的T细胞进行体外培养。我们建议分析PLX对单个T细胞亚群和它们的影响 在体内aGVHD发病机制中的意义。目标3将1)检验Dual BET和JAK1/2的假设 抑制将减少移植物抗宿主病并延长治疗反应的持续时间；以及2)评估 联合治疗对Treg和效应器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的适用性及未来临床发展 将BET和JAK1/2抑制剂结合用于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.