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Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world’s population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human–murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.

Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin lymphoma characterized by the expansion of malignant CD4+ T cells in the skin. There are two main subtypes of CTCL: an indolent form termed mycosis fungoides (MF), which is largely limited to the skin, and Sezary syndrome (SS), an aggressive leukemic variant of the disease which can manifest systemically.1-3 Previous studies have demonstrated defects in cell-mediated immunity in CTCL patients, including altered cytokine profiles and impaired neutrophil function, which lead to a high incidence of recurrent bacterial and viral infections as a result of decreased Th1-mediated immunity.4-9 It has also been reported that natural killer (NK)–cell function is decreased in CTCL patients,10-14 which could contribute to an overall decrease in the innate immune response to both neoplastic cells and viral or bacterial pathogens. Previous groups have reported that NK cells from SS patients are capable of responding to activation ex vivo, indicating the potential for development of immune-based therapeutics.15 Although MF patients often have a prolonged indolent clinical course of disease that requires localized treatment, there are few effective treatments for the successful management of patients with SS. Because of the lack of success with traditional chemotherapeutic approaches, novel immune-based therapeutics are being developed for use in a multitude of hematologic diseases, including CTCL.4,16-18 Understanding the immune microenvironment in patients with CTCL will be critical to the successful design of targeted therapies for their disease. Previous studies by our group and by others have shown increased expression of interleukin-15 (IL-15) in malignant CD4+ T cells in CTCL patients.19 IL-15 acts through a trimeric IL-15R complex to enhance NK-cell maturation and function.20-22 Indeed, in a first-in-human phase 1 trial in patients with refractory solid cancer tumors, IL-15 treatment induced profound expansion of circulating NK cells ({"type":"clinical-trial","attrs":{"text":"NCT01885897","term_id":"NCT01885897"}}NCT01885897).23 Considering that IL-15 is produced by malignant cells in CTCL, we sought to study the possible effect of chronically elevated IL-15 on NK-cell function in CTCL patients. In this study, we show that NK-cell activity is significantly enhanced in CTCL, and strikingly, higher NK-cell numbers are associated with increased mortality.

Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.

The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of FCGR3A with FCGR3B, which differ in cell type–specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a− NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B-derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a− NK cells compared with CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type–specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.

Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called "stage 3" developmental intermediate minimally characterized by a CD34(-)CD117(+)CD94(-) immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that include interleukin-1 receptor (IL-1R1)-positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here, we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes the differentiation of tonsillar IL-22-producing IL-1R1(hi) human ILC3s to CD56(bright)CD94(+) interferon (IFN)-γ-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, we demonstrate the lineage plasticity of human ILCs by identifying AHR as a transcription factor that prevents IL-1R1(hi) ILC3s from differentiating into NK cells.

It is known that microRNAs (miRs) are involved in lymphocyte development, homeostasis, activation, and occasionally malignant transformation. In this study, a miR-155 transgene (tg) was driven to be overexpressed off of the lck promoter in order to assess its effects on natural killer (NK) cell biology in vivo. miR-155 tg mice have an increase in NK-cell number with an excess of the CD11b(low)CD27(high) NK subset, indicative of a halt in terminal NK-cell differentiation that proved to be intrinsic to the cell itself. The increase in NK cells results, in part, from improved survival in medium alone and enhanced expansion with endogenous or exogenous interleukin 15. Phenotypic and functional data from miR-155 tg NK cells showed constitutive activation and enhanced target cell conjugation, resulting in more potent antitumor activity in vitro and improved survival of lymphoma-bearing mice in vivo when compared with wild type NK cells. The enhanced NK-cell survival, expansion, activation, and tumor control that result from overexpression of miR-155 in NK cells could be explained, in part, via diminished expression of the inositol phosphatase SHIP1 and increased activation of ERK and AKT kinases. Thus, the regulation of miR-155 is important for NK-cell development, homeostasis, and activation.

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.

Background: Post-transplant lymphoproliferative disease (PTLD) is a significant and often fatal complication of organ transplantation, strongly associated with Epstein-Barr virus (EBV). Severe combined immune-deficient (SCID) mice engrafted with human mononuclear cells (PBMCs) from EBV+ donors spontaneously develop human B cell lymphoproliferative disease (LPD) that resembles PTLD. Approximately 20% of EBV+ donors reproducibly develop LPD in 100% of engrafted mice (high-incidence, HI donors), and ~20% of donors never develop EBV-LPD (no-incidence, NI donors) despite all donors being EBV+. This finding suggests that host factor(s) may predispose individuals to develop EBV-LPD. PBMC from HI donors depleted of CD3+/CD4+ T helper (Th) cells do not develop EBV-LPD, suggesting that Th cells function as potential drivers of EBV-LPD. Methods: PBMCs from three HI and three NI donors were engrafted intraperitoneally into SCID mice. At week 4, spleens were harvested and human Th cells were sorted to purity (>95%). Total RNA was isolated from sorted Th cells and gene expression profiles were evaluated by RNA transcriptome analysis. Sorted Th cells from total PBMCs (resting) were used as baseline controls. Human CD45+ cells from mouse spleens were analyzed by mass cytometry (Cytometry Time of Flight, CyTOF) using a multiparametric antibody panel to identify various Th subsets. To identify specific Th subsets essential to the development of EBV-LPD, PBMCs from 3 HI donors were depleted of Th subsets: follicular Th cells (Tfh), regulatory T cells (Treg) or Tfh/Treg cells. Results: RNA Transcriptome data were analyzed using R DESeq2 for differential expression and revealed significant differences in genes associated with Tfh and Treg cell differentiation and function. Th cells sorted from mice engrafted with PBMCs from HI donors expressed high levels of IFNγ receptor, IL6R, MHC class II, FoxP3, PRMT2 and SYK genes. Applying the analysis software viSNE to CyTOF data revealed significant differences in frequency of B cells, Tfh and Treg subsets between HI and NI. Mice engrafted with Treg depleted or Tfh/Treg depleted PBMCs showed significantly improved survival compared to control nondepleted group (p-value: 0.0008, 0.0145, respectively). There was no difference in engraftment efficiency. The Tfh depleted cohort also had improved survival as compared to the control group; however, this result was not statistically significant (p-value: 0.0941). Conclusions: Significant differences in gene expression and immunophenotypic profiles exist between HI and NI donors in this spontaneous model of EBV-LPD. Depletion of Treg and Tfh subsets from PBMCs prior to engraftment significantly enhanced survival. These data suggest that select Th subsets promote EBV-LPD and provide justification for examining strategies to identify patients at risk for developing EBV-LPD in the pretransplant setting. Citation Format: Elshafa H. Ahmed, Claire Hale, Shelby Sloan, Charlene Mao, Xiaoli Zhang, H. Gulcin Ozer, Deepa Subramaniam, Frankie Jeney, Sarah Schlotter, Porsha Smith, Wing Chan, Palak Sekhri, Christoph Weigel, Christopher C. Oakes, Gregory K. Behbehani, Jianhua Yu, Mireia Guerau, Michael A. Caligiuri, Robert A. Baiocchi. Role of select T helper cell subsets in the development of Epstein-Barr virus-driven lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4691.

Among human natural killer (NK) cell intermediates in secondary lymphoid tissue (SLT), stage 3 CD34(-)CD117(+)CD161(+)CD94(-) immature NK (iNK) cells uniquely express aryl hydrocarbon receptor (AHR) and interleukin-22 (IL-22), supporting a role in mucosal immunity. The mechanisms controlling proliferation and differentiation of these cells are unknown. Here we demonstrate that the IL-1 receptor IL-1R1 was selectively expressed by a subpopulation of iNK cells that localized proximal to IL-1beta-producing conventional dendritic cells (cDCs) within SLT. IL-1R1(hi) iNK cells required continuous exposure to IL-1beta to retain AHR and IL-22 expression, and they proliferate in direct response to cDC-derived IL-15 and IL-1beta. In the absence of IL-1beta, a substantially greater fraction of IL-1R1(hi) iNK cells differentiated to stage 4 NK cells and acquired the ability to kill and secrete IFN-gamma. Thus, cDC-derived IL-1beta preserves and expands IL-1R1(hi)IL-22(+)AHR(+) iNK cells, potentially influencing human mucosal innate immunity during infection.

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56dim NK cell efficiently kills malignant targets at rest, whereas the less mature CD56bright NK cells cannot. In this study, we show that resting CD56bright NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56dim NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56bright NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell–activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell’s ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell’s PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56bright NK cell to the cytolytic CD56dim NK cells.

MicroRNAs (miRNAs) are small noncoding RNAs, 19–24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor–immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155–deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti–miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

MicroRNAs (miRs) are small, noncoding RNA molecules with important regulatory functions whose role in regulating natural killer (NK) cell biology is not well defined. Here, we show that miR-155 is synergistically induced in primary human NK cells after costimulation with IL-12 and IL-18, or with IL-12 and CD16 clustering. Over-expression of miR-155 enhanced induction of IFN-γ by IL-12 and IL-18 or CD16 stimulation, whereas knockdown of miR-155 or its disruption suppressed IFN-γ induction in monokine and/or CD16-stimulated NK cells. These effects on the regulation of NK cell IFN-γ expression were found to be mediated at least in part via miR-155's direct effects on the inositol phosphatase SHIP1. Consistent with this, we observed that modulation of miR-155 overrides IL-12 and IL-18–mediated regulation of SHIP1 expression in NK cells. Collectively, our data indicate that miR-155 expression is regulated by stimuli that strongly induce IFN-γ in NK cells such as IL-12, IL-18, and CD16 activation, and that miR-155 functions as a positive regulator of IFN-γ production in human NK cells, at least in part via down-regulating SHIP1. These findings may have clinical relevance for targeting miR-155 in neoplastic disease.

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.

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.

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.

Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses. Results: We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive (S)-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays. Conclusions: The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109

Abstract 515 The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009). Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells. Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P Thus, expression but not activity of the oncogenic product of the t(9;22) translocation is important for recruiting and allowing SET-mediated inhibition of PP2A and activation of Jak2; two events important for Ph+ stem cell survival and self renewal. Moreover, the ability of FTY720 and of its non-immunosuppressive derivatives to induce apoptosis of Ph+ progenitors and Ph+ but not normal quiescent stem cells emphasizes the notion that FTY720 and its derivatives represent strong and non-toxic anti-leukemic agents potentially useful not only for the treatment but, perhaps, for eradicating Ph+ leukemias. Disclosures: Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 3259 Poster Board III-1 We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs). First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL+ leukemias but can be extended to Jak2V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2V617F-expressing erythro-myeloid precursors revealed that Jak2V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2V617F-transduced Lin- mouse marrow progenitors. Likewise, FTY720 decreased Jak2V617F expression/activity and inhibited clonogenicity of Jak2V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner. Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34+/CD38- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice. Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL+ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction. Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex. Disclosures: Cortes: Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Histone acetylation plays a key role in the regulation of gene expression. Histone hyperacetylation is associated with chromatin opening and gene transcription, while histone hypoacetylation is associated with chromatin condensation and gene silencing. Abnormal histone hypoacetylation mediated by aberrant activity of histone deacetylases (HDACs) has been found to be associated with silencing of tumor suppressor and growth inhibitory genes in malignant cells. HDAC inhibitors (HDACIs) can relieve HDAC-mediated gene silencing and thereby induce normal patterns of cell cycle, differentiation and apoptosis in malignant cells. HDACI OSU 42 is a novel hydroxamate tethered phenylbutyrate derivative that was designed and synthesized at our institution, and exhibited IC50s at submicromolar level, compared with millimolar level for other members of this classes of HDACIs such as valproic acid (VPA). We characterized the activity of this compound in acute myeloid leukemia (AML) cells. It is known that the fusion proteins AML1/ETO and PML / RAR alpha that characterized t(8;21) and t(15;17) AML silence target genes through recruitment of HDACs to their promoter regions. Therefore we utilized AML1/ETO-positive Kasumi-1 and PML/RARA-positive NB4 cells to test the activity of HDACI OSU 42 and used THP-1 cells, characterized by AF9/MLL fusion gene, as a control. We hypothesized that by virtue of the fusion genes, Kasumi-1 and NB4 are more susceptible to HDACI treatment. IC50s for proliferation inhibition in Kasumi-1 cells treated with HDACI OSU42 were 71.8±14.3nM for 24hr and 31.3± 0.4nM for 48hr, significantly lower than VPA (2.0mM for 24hr, 0.9mM for 48hr). The IC50s for NB4 were 237.7±6.5nM for 24hr and 119±6.4nM for 48hr. As a contrast, IC50 for THP-1 was 507.3±68.3nM for 48hr. HDACI OSU42 inhibited 80% of total HDAC activity at 125nM in both Kasumi-1 and NB4; 30nM HDACI OSU42 induced hyperacetylation of histone H3 and H4. Apoptosis analysis showed that nearly 60% more of Kasumi-1 and NB4 underwent apoptosis after treated with 1μM of HDACI OSU42 for 24hr, compared with their untreated control. On the other hand, the same treatment only induced 15% more of THP-1 undergoing apoptosis. Apoptotic effect of HDACI OSU42 was mediated by activation of caspase 9 and caspase 3. Cell cycle analysis demonstrated that treatment of Kasumi-1 and NB4 with 150nM of HDACI OSU 42 inhibited cell cycle progression and arrested 20% to 30% more cells at S phase or G2/M phase, whereas this treatment had not effect on cell cycle progression of THP-1. This was consistent with the up-regulated expression of p21 at both transcription level and protein level. Q-PCR data suggested that Kasumi-1 and NB4 treated with HDACI OSU42 expressed ~10 folds of p21 higher than untreated cells. Chromatin immunoprecipitation assay revealed 10 to 50 folds increase in acetylation level of histone H3 and H4 associated with p21 promoter. Kasumi-1 and NB4 cells also show differentiation ability (increase in CD14 and CD 13 expression by flow cytometry) when treated with 30nM of HDACI OSU42, whereas THP-1 remained undifferentiated. These results support the activity of HDACI OSU42 as a new potent HDACI in AML.

We developed a mouse model of acute T/natural killer (NK) acute lymphoblastic leukemia (ALL) that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We demonstrated the non-random nature of methylation in the transformation from benign polyclonal expansion to leukemia, and identified a novel tumor suppressor gene silenced in the majority of human leukemia but not solid tumors. To this, we utilized restriction landmark genomic scanning (RLGS) on eight IL-15 transgenic (tg) mice with T/NK ALL, and performed RLGS on an additional five spleens from IL-15tg mice without ALL, but with polyclonal T/NK expansion, and on four spleens from wild type (wt) mice. A total of 2447 RLGS fragments with good resolution on each RLGS profile were analyzed. Only 1–2 variable changes were detected in either wt or polyclonal T/NK expansion controls. In contrast, the eight spleens with clonal T/NK leukemia had 45 to 209 changes (1.8 to 8.5% of total fragments) consistent with aberrant DNA methylation. The association of RLGS fragment losses or reduced intensity with leukemic transformation versus polyclonal expansion was highly significant (P