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The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

Posttranscriptional modification of histones by methylation plays an important role in regulating Ag-driven T-cell responses. We have recently drawn correlations between allogeneic T-cell responses and the histone methyltransferase Ezh2, which catalyzes histone H3 lysine 27 trimethylation. The functional relevance of Ezh2 in T-cell alloimmunity remains unclear. Here, we identify a central role of Ezh2 in regulating allogeneic T-cell proliferation, differentiation, and function. Conditional loss of Ezh2 in donor T cells inhibited graft-versus-host disease (GVHD) in mice after allogeneic bone marrow (BM) transplantation. Although Ezh2-deficient T cells were initially activated to proliferate upon alloantigenic priming, their ability to undergo continual proliferation and expansion was defective during late stages of GVHD induction. This effect of Ezh2 ablation was largely independent of the proapoptotic molecule Bim. Unexpectedly, as a gene silencer, Ezh2 was required to promote the expression of transcription factors Tbx21 and Stat4. Loss of Ezh2 in T cells specifically impaired their differentiation into interferon (IFN)-γ–producing effector cells. However, Ezh2 ablation retained antileukemia activity in alloreactive T cells, leading to improved overall survival of the recipients. Our findings justify investigation of modulating Ezh2 as a therapeutic strategy for the treatment of GVHD and other T cell–mediated inflammatory disorders.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies, however, it remains to be limited by morbidity and mortality related to graft-versus-host disease (GVHD). During GVHD, donor T cells are activated by host antigen-presenting cells (APCs) and differentiate into effector cells that mediate host tissue damage. Development of alloreactive effector T cells requires orchestrated expression of myriad genes initiated by host APCs. However, little is known about the key epigenetic factor that controls this process. Histone methylation, which is catalyzed by histone methyltransferanses (HMT), has been correlated with genes encoding effector cytokines and transcription factors critical for effector differentiation. We recently demonstrated that inhibition of histone methylation using 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD through induction of apoptosis in alloreactive T cells. However, since DZNep reduced multiple histone methylation marks, including trimethylation of histone H3 at lysine 4 (H3K4me3), H3K27me3, H3K36me3 and H4K20me3, the key HMT that regulates the transcription program important for allogeneic T cell responses remains unclear. Using genetic approaches and mouse GVHD models, we identify that the HMT Ezh2, which specifically catalyzes H3K27me3 and acts primarily as a gene silencer, plays a central role in regulating allogeneic T cell proliferation, differentiation, and function during the GVHD process. Transfer of donor T cells derived from Ezh2 conditional knockout C57BL/6 (B6) mice did not induce GVHD in lethally irradiated allogeneic BALB/C mice, with all recipients surviving free of disease. In contrast, wild-type (WT) donor B6 T cells caused uniformly lethal GVHD in these recipients. We found that loss of Ezh2 selectively impaired alloantigen-specific T cell responses. Three days after allo-HSCT, both Ezh2-deficient and WT T cells were similarly activated to proliferate in vivo. However, 7 days after transplantation, there was a significant reduction in the number of highly proliferative Ezh2-deficient T cells compared to WT T cells, leading to a dramatic reduction of infiltrating Ezh2-deficient T cells in GVHD target organs. Interestingly, conditionally deleting Ezh2 did not affect proliferation and survival of B6 T cells that were transferred into lethally irradiated syngeneic B6/SJL mice by 7 days after transplantation. Thus, Ezh2 is critical for the continual proliferation of alloantigen-responding T cells during later stages of GVHD induction. In contrast, Ezh2 is dispensable for both the initial activation and proliferation of T cells upon alloantigen-priming and homeostatic expansion of T cells in response to lymphopenia. Previous studies suggest that IFN-g-producing alloreactive effector cells cause damage to the liver and gastrointestinal tract. We found that loss of Ezh2 led to selective reduction in frequency of IFN-γ-producing effector cells 7 days after allo-HSCT. This effect was accompanied with decreased expression of transcription factors TBX21 and STAT4, both of which are crucial for effector differentiation. These data suggest that in addition to regulating the transcription program critical for producing sufficient numbers of alloreactive effector T cells needed to mediate GVHD, Ezh2 is also important for orchestrating genes required to promote IFN-γ production in effector cells. Importantly, we found that Ezh2 ablation retained anti-leukemia activity in alloreactive T cells, leading to improved overall survival of transplant recipients. Collectively, these findings identify Ezh2 as a key epigenetic regulator that controls allogeneic T cell responses and GVHD. We propose that pharmacological modulation of Ezh2 using specific inhibitors should be investigated as a novel therapeutic strategy after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 953 Adoptive T cell therapy has the potential to enhance antitumor immunity and improve vaccine efficacy, of which a key challenge is to generate sufficient numbers of T cells that can persist in vivo after transfer. Cellular metabolism plays important roles in regulating T cell proliferation and survival. T cells responding to antigen activation dramatically upregulate both glycolysis and oxidative phosphorylation (OXPHOS), leading to increased production of adenosine triphosphate (ATP) and metabolic intermediates that are required for cell growth and proliferation. Without sufficient support for their demands, activated T cells may be deleted or become quiescent. Thus, better understanding of the mechanism that regulates cellular metabolism in T cell response will lead to new strategies to improve the efficacy of adoptive T cell therapy. Here we explore the functional impact of an epigenetic pathway in cellular metabolism in antigen-driven T cells and tumor immunity. Using genetic approaches and experimental mouse models, we demonstrate that Ezh2, which is a histone methyltransferase that represses the transcription of cohorts of developmental regulators, promotes the survival and expansion of antigen-driven T cells through regulating bioenergetic metabolism. Conditional deletion of Ezh2 caused selective apoptosis in T cells upon activation with alloantigens in vivo and in vitro or with T cell receptor (TCR)-ligation in vitro. Ezh2 deficiency resulted in markedly increased expression of proapoptotic gene Bim, but had no significant impact on the expression of other Bcl-2 family members (e.g., anti-apoptotic genes Bcl-2 and Bcl-xL,). Genetic inactivation of Bim only slightly improved the survival of alloantigen-activated Ezh2-deficient T cells, suggesting that Ezh2 may control T-cell immunity largely through a Bim-independent mechanism. This differs from our recent observations showing that Bim is required for increased apoptosis in activated T cells treated with a pharmacologic inhibitor of Ezh2 and histone methylation 3-Deazaneplanocin A (Blood, 2012). Our prior studies and others suggest that impaired cellular metabolism may lead to increased apoptosis of antigen-activated T cells. We observed that upon TCR-ligation Ezh2 null T cells were incapable to upregulate OXPHOS as compared to wild-type (WT) T cells, which was accompanied with reduced ATP levels and increased reactive oxygen species (ROS). Neutralization of ROS by N-acetylcysteine significantly improved the survival of TCR-activated Ezh2 null T cells. Interestingly, overexpression of WT Ezh2 in TCR-activated Ezh2 null T cells, but not enzymatically inactive H689A Ezh2 mutant or nuclear localization-inactive Ezh2 mutant, restored the ability of Ezh2-deficient T cells to upregulate OXPHOS, reduced ROS levels, and rescued their survival capability in vitro. These results suggest that Ezh2 is important for regulating bioenergetic metabolism in activated T cells. Furthermore, the nuclear but not cytoplasmic Ezh2 is required to regulate bioenergetic metabolism in activated T cells during clonal expansion phase, although Ezh2 in the cell cytoplasm could be involved in regulating actin polymerization. In mouse models of graft-versus-host disease (GVHD) and leukemia, transfer of donor T cells lacking Ezh2 failed to mediate GVHD and anti-leukemia activity in mice receiving allogeneic bone marrow transplantation. In addition, Ezh2 deficiency also ablated the ability of adoptively transferred antigen-specific CD8 T cells to control tumor growth in mice with established melanoma. Importantly, the absence of Ezh2 did not impair the development of effector T cells producing IFN-γ, granzyme B, Fas ligand and Trail, ruling out the possibility that impaired T-cell immunity of Ezh2 null T cells results from defective effector differentiation. Our findings identify the critical role of Ezh2 in regulating bioenergetic metabolism in antigen-driven T cells, therefore for the first time linking the epigenetic pathway to cellular metabolism in T cell response. Thus, Ezh2 and its-regulated bioenergetic metabolism may represent novel targets to improve the efficacy of adoptive T-cell immunotherapy. Modulation of Ezh2 and its activity may have broad implications in the treatment of many other inflammatory disorders, such as graft rejection after organ transplantation, GVHD and autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.