Ezh2-mediated Epigenetic Effects and Alloimmunity

 描述(申请人提供)：移植物抗宿主病(GVHD)仍然是造血干细胞移植(allo-HSCT)后发病率和死亡率的主要原因，发生在30-70%的移植后，占死亡的15%。急性GVHD以宿主组织损伤为特征，供体T细胞与供体或宿主来源的抗原提呈细胞(APC)相互作用后，由供体T细胞介导。APC和同种异体抗原反应性供体T细胞之间的相互作用导致转录因子的丰度和功能的变化，进而刺激或抑制同种异体反应性T细胞反应和GVHD关键基因的表达。表观遗传改变被认为在调节同种异体反应性T细胞的基因表达方面很重要，但表观遗传过程如何通过影响基因转录来帮助调节同种异体反应性T细胞的反应在很大程度上仍不清楚。我们的长期目标是确定关键的表观遗传调控因子(S)，可以靶向调节GVHD。本应用的目的是研究组蛋白甲基转移酶Ezh2如何协调转录因子1的表达，并确定在GVHD中调节Ezh2的最佳药理学方法。组蛋白甲基转移酶主要起基因沉默的作用。我们之前发现，Ezh2功能的遗传失活降低了GVHD，但保留了移植物抗白血病(GVL)反应，从而提高了接受allo-HSCT治疗的白血病小鼠的存活率。这些发现形成了一个中心假设：A)Ezh2是同种异体反应性T细胞反应的主要调节因子，B)靶向Ezh2可能导致新的和临床相关的策略来调节GVHD。出乎意料的是，我们的初步研究表明，新发现的可以选择性降低H3K27me3的Ezh2抑制剂，未能控制小鼠的GVHD。因此，针对Ezh2的最佳方法仍然是控制GVHD的一个未得到满足的需求。最近，我们发现：1)Ezh2促进同种异体反应性T细胞转录因子T-bet和ID3的表达，T-bet是T辅助细胞(Th)1细胞发育所必需的，ID3是效应细胞存活和记忆细胞形成所必需的。在移植物抗宿主病诱导的后期阶段，Ezh2的缺失导致产生干扰素-γ的同种异体反应性T细胞的增殖和存活受到选择性的损害。Ezh2的这种激活功能与其 2)Ezh2 SET结构域，它包含一个酶单位，正向调节T细胞中Ezh2蛋白的稳定性和功能。缺乏SET结构域的突变Ezh2蛋白被蛋白酶体迅速降解，无法与Hsp90形成稳定的复合体，Hsp90是一种帮助稳定蛋白质表达和功能的伴侣蛋白。Hsp90的药理抑制可导致GVHD小鼠T细胞受体激活的T细胞中Ezh2蛋白迅速降解，同种异体T细胞反应明显降低。值得注意的是，尽管Hsp90有几个关键的信号中间产物，但Ezh2的过表达拯救了激活的T细胞，使其免于Hsp90抑制诱导的凋亡。这表明，通过抑制Hsp90来耗尽Ezh2蛋白可能是一种在同种异体T细胞中靶向Ezh2的新方法；3)在人类中，通过靶点水平评估的EZH2活性，在GVHD发生时来自allo-HSCT接受者的T细胞比来自非GVHD患者的T细胞更高。我们的初步发现表明，EZH2在介导aGVHD患者的人T细胞对钙调神经磷酸酶抑制剂耐药性方面具有重要作用，这种作用可以用流式细胞仪分析来量化。这为这一表观遗传机制在人类中的相关性提供了实质性证据，并增强了Ezh2调制的翻译潜力。为了进一步详细检验我们的中心假设，我们将确定ID3和T-bet在介导Ezh2调节allo-HSCT后小鼠同种异体T细胞反应中的关键作用；确定靶向Ezh2-Hsp90复合体对GVH反应的影响；并表征EZH2介导的T细胞在人类GVHD中的功能，并建立其作为GVHD生物标志物的实用性。这些研究可能会导致：A)针对Ezh2激活功能的新的临床相关策略，以提高allo-HSCT的疗效；以及B)更好地理解同种异体反应性T细胞的表观遗传效应及其对T细胞免疫的调节。

 DESCRIPTION (provided by applicant): Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after hematopoietic stem cell transplantation (allo-HSCT), occurring after 30-70% of transplants and accounting for up to 15% of deaths. Acute GVHD is characterized by host tissue injury, mediated by donor T cells following interaction with either donor- or host-derived antigen presenting cells (APCs). The interaction between APCs and alloantigen-responsive donor T-cells leads to changes in abundance and function of transcription factors, which in turn stimulate or repress expression of genes crucial for alloreactive T cell responses and GVHD. Epigenetic changes are thought to be important in regulation of gene expression in alloreactive T-cells, but it remains largely unknown how epigenetic processes help regulate alloreactive T cell responses by affecting gene transcription. Our long- term goal is to identify key epigenetic regulator(s) that can be targeted to modulate GVHD. The objective of this application is to investigate how Ezh2, a histone methyltransferase that acts primarily as a gene silencer, orchestrates the expression of transcription factors criticl for alloreactive T cell responses and identify an optimal pharmacological approach to modulate Ezh2 in GVHD. We previously discovered that genetic inactivation of Ezh2 function reduced GVHD but preserved graft- versus-leukemia (GVL) responses, leading to improved survival of mice with leukemia treated with allo-HSCT. These findings form a central hypothesis that: A) Ezh2 is a master regulator of alloreactive T-cell responses, and B) targeting Ezh2 may lead to novel and clinically relevant strategies to modulate GVHD. Unexpectedly, our preliminary studies showed that newly discovered Ezh2 inhibitors that can selectively reduce H3K27me3, failed to control GVHD in mice. Thus, optimal methods to target Ezh2 remain an unmet need for controlling GVHD. Most recently, we identified that: 1) Ezh2 promoted the expression of transcription factors T-bet (which is essential for development of T helper (Th)1 cells) and Id3 (which is important for effector cell survival and memory cell formation) in alloreactive T cells. Loss of Ezh2 led to selectively impaired expansion and survival of alloreactive T cells producing IFN-γ during late stages of GVHD induction. This activation function of Ezh2 is in contrast to its previously described role as a gene silencer; 2) the Ezh2 SET domain, which contains an enzymatic unit, positively regulated Ezh2 protein stability and function in T cells. Mutant Ezh2 protein that lacked the SET domain was rapidly degraded by proteasomes, and failed to form a stable complex with Hsp90, a chaperone protein that helps stabilize protein expression and function. Pharmacological inhibition of Hsp90 resulted in rapid degradation of Ezh2 protein in T cell receptor-activated T cells and marked decrease of allogeneic T cell responses in GVHD mice. Notably, although Hsp90 has several key signaling intermediates, overexpression of Ezh2 rescued activated T cells from Hsp90 inhibition-induced apoptosis. This indicates that depleting Ezh2 protein by Hsp90 inhibition may represent a novel approach to target Ezh2 in allogeneic T cells; and 3) in humans, EZH2 activity, assessed by levels of its targets, was higher in T cells from allo-HSCT recipients at the time of GVHD onset compared to T cells from patients without GVHD. Our preliminary findings point to the importance of EZH2 in mediating calcineurin inhibitor-resistance in human T cells from aGVHD patients, and this effect can be quantified using flow cytometric analysis. This provides substantial evidence for the relevance of this epigenetic mechanism in humans and strengthens the translational potential of Ezh2 modulation. To further test our central hypothesis in detail, we will determine the critical roles f Id3 and T-bet in mediating Ezh2 regulation of allogeneic T cell responses in mice after allo-HSCT; determine the effects of targeting the Ezh2-Hsp90 complex on GVH responses; and characterize EZH2-mediated T-cell function in human GVHD and establish its utility as a GVHD biomarker. These studies would potentially lead to: A) novel and clinically relevant strategies to target Ezh2 activation function for improving the efficacy of allo-HSCT; and B) improved understanding of the epigenetic effects in alloreactive T cells and its regulation of T cell immunity.