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EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses., Author summary Epstein-Barr virus (EBV), one of the human gamma-herpesviruses, is a well-defined viral agent that strongly associates with malignancies of lymphoid and epithelial origin. EBV-associated gastric carcinoma (EBVaGC) is the most common malignancy caused by EBV infection. In this paper, we identified that beyond tumor suppressor genes, immune related genes (IRGs) are another group of host genes that are extensively DNA hypermethylated and epigenetically silenced in EBVaGC comparing to non-EBV GC. For certain IRGs, such as metallothionein-1 (MT1) and homeobox A (HOXA) genes, the whole gene clusters undergo DNA hypermethylation in EBVaGC, while it also occurs for isolated individual IRGs, such as IRAK2 and MAL. Considering the critical role of innate immunity in antiviral control, we expected that silencing of IRGs would benefit EBV viral replication and propagation. Indeed, our further investigation of several IRGs (MT1G, HOXA10, IRAK2, and MAL) confirmed their antiviral activities against EBV lytic replication. Some of them also suppress lytic replication of another human gamma-herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV), indicating their broad antiviral spectrum. Collectively, our results demonstrated that epigenetic silencing of antiviral IRGs is an efficient viral strategy utilized by oncoviruses, such as EBV, to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis.

Introduction. Our previous flow cytometry-based comparison of HIV(+) and HIV(-) autologous hematopoietic stem cell transplant (auto-HSCT) recipient immunomes at 56, 180 and 365 days post-transplant to each other and to healthy controls (HCs) showed that both sets of auto-HSCT recipient immunomes approached HCs over time, but retained significant differences. HIV(+), but not HIV(-), auto-AHCT recipients retained pro-inflammatory features consistent with chronic HIV infection. Here, we report the results of a quantitative and functional analysis of immune reconstitution in HIV(+) patients treated with allogeneic hematopoietic stem cell transplant (allo-HSCT), in comparison with HIV(+) auto-HSCT recipients and HCs. Methods. Blood samples were collected for analysis at days 56, 180 and 365 post-transplant from HIV(+) transplant recipients and at 1 time point from HCs. Whole blood analysis was performed by five-color flow cytometry across 100 immune marker combinations. Comparisons were made between HIV(+) allo-HSCT recipients (n=17, acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, Hodgkin and non-Hodgkin lymphoma that received myeloablative or reduced intensity conditioning on the BMT-CTN-0903/AMC-080 trial), HIV(+) auto-HSCT recipients (n=36, aggressive B cell non-Hodgkin lymphoma or Hodgkin lymphoma that received myeloablative conditioning on the BMT-CTN-0803/AMC-071 trial) and 71 HCs. Unsupervised principal component analysis (PCA) examined differences in immune cell proportions, identified by flow cytometry across 100 cell subsets at each time point. Wilcoxon rank-sum tests compared median absolute counts and median proportions of cell subsets. An independent feature importance score analysis (FIS) identified contributions of immune cell populations expressing specific immune marker combinations to the differences between HIV(+) auto-HSCT recipients, HIV(+) allo-HSCT recipients and HCs. Functional responsiveness of HIV(+) allo-HSCT recipients' T cells to stimulation with CD3- and CD28-directed antibodies, NK cells to stimulation with IL-12 and IL-18 and monocytes to stimulation with lipopolysaccharide (LPS) was assessed in a preliminary mass cytometry on peripheral blood mononuclear cells isolated at the same time points (n=2) and compared to HCs (n=2). Results. PCA showed that immunomes of HIV(+) allo-HSCT recipients and HIV(+) auto-HSCT recipients clustered together with each other, but away from HCs at all time points throughout the post-transplant year. FIS identified: 1) 13 cell subsets that defined the difference between HIV(+) allo-HSCT recipients (all visits) and HCs, and 2) 11 immune cell subsets that defined the difference between HIV(+) auto-HSCT recipients (all visits) and HCs; in both of these comparisons, activated CD3+/HLA-DR+ T cells had the greatest impact on the difference between HIV(+) and HC immunomes. At 1 year, both HIV(+) transplant recipient cohorts had higher absolute numbers of activated T cells, effector T cells and CD8+ T cells than HCs (Wilcoxon rank-sum test, p Conclusion. Chronic HIV infection confers the pro-inflammatory immune features on the phenotypic and functional profiling of the T lymphocyte immunome of stem cell transplant recipients, irrespective of allogeneic or autologous stem cell donor source. Disclosures Devine: Bristol Myers: Other: Grant for monitoring support & travel support; Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Noy:Janssen: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria; NIH: Research Funding; Pharamcyclics: Research Funding; Raphael Pharma: Research Funding. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Hofmeister:Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Navarro:Atara Biotherapeutics: Employment, Equity Ownership. Behbehani:Fluidigm corporation: Other: Travel funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding. Baiocchi:Prelude: Consultancy.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and Flt3-ITD. MllPTD/WT:Flt3ITD/WT mice developed acute leukemia with 100% penetrance, at a median of 49 weeks. As in human MLL-PTD and/or the FLT3-ITD AML, mouse blasts exhibited normal cytogenetics, decreased Mll-WT-to-Mll-PTD ratio, loss of the Flt3-WT allele, and increased total Flt3. Highlighting the adverse impact of FLT3-ITD dosage on patient survival, mice with homozygous Flt3-ITD alleles, MllPTD/WT:Flt3ITD/ITD, demonstrated a nearly 30-week reduction in latency to overt AML. Here we demonstrate, for the first time, that Mll-PTD contributes to leukemogenesis as a gain-of-function mutation and describe a novel murine model closely recapitulating human AML.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world's population and is linked with cancer development. In immune-competent individuals, EBV-infection is controlled by a highly efficient virus-specific T cell response. Following primary infection, the virus achieves lifelong persistence within the human host. Risk of EBV-driven cancers increases with immune suppression (IS). Solid organ transplant recipients receive IS medications to prevent graft rejection and are at highest risk of developing EBV-associated lymphomas known as post-transplant lymphoproliferative disease (PTLD). PTLD represents a serious complication of organ transplantation, associated with poor prognosis. Currently, no standard approach for prevention or treatment exists. Reducing the level of IS medication may control PTLD but often leads to graft-rejection. In order to promote long-term protection from EBV-driven cancers, we have developed a vaccine to bolster EBV-specific immunity by targeting the EBV immediate early protein, BZLF1. BZLF1 initiates the activation of lytic stage in EBV-infected cells and promotes B-cell transformation. Work by our group has shown BZLF1-specific T cell expansion following reduction in IS medications correlates with PTLD tumor regression and improved patient survival. Here we specifically delivered the protein (BZLF1) to dendritic cells (DCs) through its endocytic receptor DEC205. DCs were generated from HLA-B8+ donor monocytes incubated with interleukin-4 (IL4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Mature DCs were then loaded with DEC205-BZLF1 fusion protein or control protein (DEC205-Human Chorionic Gonadotropin (DEC205-HCG)). Antigen-loaded DCs were co-cultured with autologous peripheral blood mononuclear cells (PBMCs) in the presence of IL-2 for 10 days. Cells were analyzed by flow cytometry using HLA-tetramers to detect and quantify antigen-specific cytotoxic T leukocyte (CTL) response. To test the EBV vaccine in-vivo, we utilized a human-murine chimeric model of EBV-driven lymphoproliferative disease (EBV-LPD). Severe combined immune deficient (SCID) mice were engrafted with PBMCs from EBV+ donors (Hu-PBL-SCID model). The spontaneous EBV-LPD that develops in this model is comprised of human CD20+, EBV+ lymphoblasts that closely resembles PTLD. Mice were immunized with DCs loaded with DEC205-BZLF1 or DEC205-HCG at the time of PBMC transplant and received booster doses at day 14 and 28. Splenocyte from vaccinated mice were stimulated with autologous tumor (lymphoblastoid cells line, (LCL)) pulsed with BZLF1 pepmix, BZLF1 pepmix alone, and anti-CD3. Secretion of IFNg by stimulated splenocytes was detected using Human IFNg Enzyme-Linked Immunosorbent Spot (ELISpot). In vitro co-cultures treated with DEC205-BZLF1-loaded DCs showed increased expansion of EBV-specific CTLs (p-value: 0.0002) capable of abundant IFNg production and potent cytotoxicity against autologous tumor. This vaccine significantly improved survival in vaccinated mice (p-value: 0.035). Splenocytes from mice in the DEC205-BZLF1 vaccination group revealed higher responsiveness to autologous LCLs and BZLF1 pepmix compared to controls as determined by ELISpot. Human cells recovered from mouse spleen will be analyzed by mass cytometry using a multi-parametric antibody panel to evaluate central/effector memory status, CD4+ Th, CD8+ CTL, NK, and monocyte subsets. These results further support pre-clinical and clinical development of vaccine approaches utilizing the BZLF1 protein as an immunogen to harness adaptive cellular responses to prevent EBV-associated LPD in vulnerable patient populations. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a devastating disease primarily affecting adults. Immune evasion is a major mechanism of AML persistence, and represents a barrier for long-term clinical success. Natural killer (NK) cells are a key component of the innate immune system and hold promise as a tool for effective anti-leukemia therapy. However, to date, the clinical success of NK therapy has been disappointing, indicating additional immune evasion strategies may affect the ability of NK cells to function in patients. We hypothesized that AML may evade the innate immune system by inhibiting NK cell maturation. To evaluate NK function and maturation during AML progression, our lab utilized a novel knock-in model of AML that expresses both Flt3-ITD and Mll-PTD mutations (PTD/ITD), and develops AML with 100% penetrance and recapitulates human disease. For these studies, both primary PTD/ITD mice and transplanted AML blasts were used. For transplant studies, AML or wild-type (WT) control cells (CD45.2+) were transplanted into irradiated C57BL/6 (CD45.1+) naïve recipients. Normal (CD45.1+) NK cells were defined as NK1.1+/CD3- by flow cytometry, and expression of receptors was determined within this population. NK cells in leukemic mice exhibited a reduction in the activating receptors Ly49D and Ly49H in the spleen, blood, LN, and bone marrow. There was also an increase in the inhibitory receptor Ly49C/I and NKG2C/A/E (both p NK maturation was then evaluated by examining CD11b and CD27 protein expression on NK1.1+/CD3- cells in the spleen, bone marrow, blood, and LN. CD11b+/CD27+ (DP) NK cells were reduced in the spleen as well as bone marrow, blood and LN (p Disclosures No relevant conflicts of interest to declare.