Ezh2-mediated Epigenetic Effects and Alloimmunity

 描述（由申请方提供）：移植物抗宿主病（GVHD）仍然是造血干细胞移植（allo-HSCT）后发病和死亡的主要原因，发生在30-70%的移植后，占死亡的15%。急性GVHD的特征是宿主组织损伤，由供体T细胞与供体或宿主来源的抗原呈递细胞（APC）相互作用后介导。APC和同种异体抗原应答供体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，这反过来刺激或抑制对同种异体反应性T细胞应答和GVHD至关重要的基因的表达。表观遗传变化被认为是重要的基因表达的调节同种异体反应性T细胞，但它仍然在很大程度上是未知的表观遗传过程如何帮助调节同种异体反应性T细胞反应，通过影响基因转录。我们的长期目标是确定关键的表观遗传调节因子，可以有针对性地调节GVHD。本申请的目的是研究Ezh 2（主要作为基因沉默物的组蛋白甲基转移酶）如何协调对于同种异体反应性T细胞应答至关重要的转录因子1的表达，并鉴定在GVHD中调节Ezh 2的最佳药理学方法。我们先前发现Ezh 2功能的遗传失活减少了GVHD，但保留了移植物抗白血病（GVL）应答，导致用allo-HSCT治疗的患有白血病的小鼠的存活率提高。这些发现形成了一个中心假设：A）Ezh 2是同种异体反应性T细胞应答的主要调节剂，和B）靶向Ezh 2可能导致调节GVHD的新的和临床相关的策略。出乎意料的是，我们的初步研究表明，新发现的Ezh 2抑制剂，可以选择性地减少H3 K27 me 3，未能控制小鼠GVHD。因此，靶向Ezh 2的最佳方法仍然是控制GVHD的未满足的需求。最近，我们发现：1）Ezh 2促进同种异体反应性T细胞中转录因子T-bet（其对于T辅助（Th）1细胞的发育是必需的）和Id 3（其对于效应细胞存活和记忆细胞形成是重要的）的表达。Ezh 2的缺失导致在GVHD诱导的晚期阶段产生IFN-γ的同种异体反应性T细胞的选择性受损的扩增和存活。Ezh 2的这种激活功能与其 先前描述的作为基因沉默子的作用; 2）Ezh 2 SET结构域，其含有酶单位，正调控Ezh 2蛋白在T细胞中的稳定性和功能。缺乏SET结构域的突变Ezh 2蛋白被蛋白酶体迅速降解，并且未能与Hsp 90形成稳定的复合物，Hsp 90是一种有助于稳定蛋白表达和功能的伴侣蛋白。Hsp 90的药理学抑制导致T细胞受体激活的T细胞中Ezh 2蛋白的快速降解和GVHD小鼠中同种异体T细胞应答的显著降低。值得注意的是，虽然Hsp 90有几个关键的信号传导中间体，Ezh 2的过度表达拯救活化的T细胞从Hsp 90抑制诱导的凋亡。这表明，通过Hsp 90抑制耗尽Ezh 2蛋白可能代表了在同种异体T细胞中靶向Ezh 2的新方法;和3）在人中，与来自没有GVHD的患者的T细胞相比，在GVHD发作时来自allo-HSCT接受者的T细胞中的EZH 2活性（通过其靶标的水平评估）更高。我们的初步发现指出EZH 2在介导aGVHD患者的人T细胞中钙调磷酸酶介导物抗性中的重要性，并且这种作用可以使用流式细胞术分析来量化。这为这种表观遗传机制在人类中的相关性提供了大量证据，并加强了Ezh 2调节的翻译潜力。为了进一步详细检验我们的中心假设，我们将确定Id 3和T-bet在allo-HSCT后介导小鼠中同种异体T细胞应答的Ezh 2调节中的关键作用;确定靶向Ezh 2-Hsp 90复合物对GVH应答的影响;并表征Ezh 2介导的T细胞在人GVHD中的功能并建立其作为GVHD生物标志物的效用。这些研究将潜在地导致：A）靶向Ezh 2活化功能以改善allo-HSCT的功效的新颖且临床相关的策略;和B）改善对同种异体反应性T细胞中的表观遗传效应及其对T细胞免疫的调节的理解。

 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.