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-? 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.