Siglec-6 as a therapeutic target for cell migration and adhesion in chronic lymphocytic leukemia.

Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.

Follicular Helper and Regulatory T Cells Drive the Development of Spontaneous Epstein-Barr Virus Lymphoproliferative Disorder.

Epstein-Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention.

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

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.

Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development.

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.

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

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.

Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells.

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.

PTEN is a negative regulator of NK cell cytolytic function.

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 CD56(dim) NK cell efficiently kills malignant targets at rest, whereas the less mature CD56(bright) NK cells cannot. In this study, we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56(bright) 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 CD56(bright) NK cell to the cytolytic CD56(dim) NK cells.

The transcription Factor AHR prevents the differentiation of a stage 3 innate lymphoid cell subset to natural killer cells.

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.

Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer 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.

MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response.

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.

Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias.

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. Mll(PTD/WT):Flt3(ITD/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, Mll(PTD/WT):Flt3(ITD/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.

Regulation of acute graft-versus-host disease by microRNA-155.

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.

miR-155 regulates IFN-γ production in natural killer cells.

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.

Interleukin-1beta selectively expands and sustains interleukin-22+ immature human natural killer cells in secondary lymphoid tissue.

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.

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia.

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia 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 used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.

A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies.

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.