Harnessing natural killer cell effector function against cancer.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that participate in anti-tumour and anti-viral immune responses. Their ability to rapidly destroy abnormal cells and to enhance the anti-cancer function of dendritic cells, CD8+ T cells, and macrophages makes them an attractive target for immunotherapeutic strategies. The development of approaches that augment NK-cell activation against cancer is currently under intense preclinical and clinical research and strategies include chimeric antigen receptor NK cells, NK-cell engagers, cytokines, and immune checkpoint inhibitors. In this review, we highlight recent advances in NK-cell therapeutic development and discuss their potential to add to our armamentarium against cancer.

XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression.

The first-in-class inhibitor of exportin-1 (XPO1) selinexor is currently under clinical investigation in combination with the BTK inhibitor ibrutinib for patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma. Selinexor induces apoptosis of tumour cells through nuclear retention of tumour suppressor proteins and has also recently been described to modulate natural killer (NK) cell and T cell cytotoxicity against lymphoma cells. Here, we demonstrate that XPO1 inhibition enhances NK cell effector function against primary CLL cells via downregulation of HLA-E and upregulation of TRAIL death receptors DR4 and DR5. Furthermore, selinexor potentiates NK cell activation against CLL cells in combination with several approved treatments; acalabrutinib, rituximab and obinutuzumab. We further demonstrate that lymph node associated signals (IL-4 + CD40L) inhibit NK cell activation against CLL cells via upregulation of HLA-E, and that inhibition of XPO1 can overcome this protective effect. These findings allow for the design of more efficacious combination strategies to harness NK cell effector functions against CLL.

Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL.

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells' innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3'UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

Targeting KIR as a novel approach to improve CAR-NK cell function.

Chimeric antigen receptor (CAR) NK cells are demonstrating promising activity in clinical trials and possess a favorable safety profile compared to CAR-T cells. The Killer cell Immunoglobulin-like Receptors (KIR) have a critical role in the control of NK cell function, and recently, this family of activating and inhibitory receptors have been targeted to improve CAR-NK function. These strategies include the utilisation of inhibitory KIR to reduce trogocytosis-associated NK cell fratricide, the downregulation of inhibitory KIR on CAR-NK cells to alleviate HLA mediated suppression, the selection of CAR-NK cell donors enriched for activating KIR, and the use of activating KIR intracellular domains within novel CAR constructs. These pre-clinical studies demonstrate the potential utility of targeting the KIR to improve CAR-NK cell efficacy and patient outcomes.

Disruption of the NKG2A:HLA-E Immune Checkpoint Axis to Enhance NK Cell Activation against Cancer.

Ligation of the inhibitory receptor NKG2A by its ligand HLA-E negatively regulates the activation of natural killer (NK) cells, as well as subsets of CD8+ T cells and innate T cell populations. NKG2A has recently become a novel immune checkpoint target for the treatment of cancer and direct antibody mediated blockade of NKG2A function is currently under assessment in two phase 3 clinical trials. In addition to direct targeting, the NKG2A:HLA-E axis can also be disrupted indirectly via multiple different targeted cancer agents that were not previously recognised to possess immunomodulatory properties. Increased understanding of immune cell modulation by targeted cancer therapies will allow for the design of rational and more efficacious drug combination strategies to improve cancer patient outcomes. In this review, we summarise and discuss the various strategies currently in development which either directly or indirectly disrupt the NKG2A:HLA-E interaction to enhance NK cell activation against cancer.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

Selinexor Enhances NK Cell Activation Against Malignant B Cells via Downregulation of HLA-E.

Selinexor is an FDA approved selective inhibitor of the nuclear export protein exportin-1 (XPO1) and causes specific cancer cell death via nuclear accumulation of tumor suppressor proteins. Design of rational studies for the use of selinexor in combination with other therapeutic agents, such as immunotherapies, requires a fundamental understanding of the effects of selinexor on the immune system. One important emerging area of immunotherapy are natural killer (NK) cell based therapeutics. NK cell function is tightly regulated by a balance of signals derived from multiple activating and inhibitory receptors. Thus in cancer, up-regulation of stress ligands recognised by activating receptors or down-regulation of HLA class I recognised by inhibitory receptors can result in an anti-cancer NK cell response. Changes in XPO1 function therefore have the potential to affect NK cell function through shifting this balance. We therefore sought to investigate how selinexor may affect NK cell function. Selinexor pre-treatment of lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4, JeKo-1 and Ramos cells, concurrent with increased CD107a and IFNγ expression on NK cells. In addition, selinexor enhanced ADCC against lymphoma cells coated with the anti-CD20 antibodies rituximab and obinutuzumab. In probing the likely mechanism, we identified that XPO1 inhibition significantly reduced the surface expression of HLA-E on lymphoma cell lines and on primary chronic lymphocytic leukemia cells. HLA-E binds the inhibitory receptor NKG2A and in accordance with this, selinexor selectively increased activation of NKG2A+ NK cells. Our data reveals that selinexor, in addition to its direct cytotoxic activity, also activates an anti-cancer immune response via disruption of the inhibitory NKG2A:HLA-E axis.

The role of NK cells in oncolytic viral therapy: a focus on hepatocellular carcinoma.

Natural killer (NK) cells have a key role in host anti-tumour immune responses via direct killing of tumour cells and promotion of adaptive immune responses. They are therefore attractive targets to promote the anti-tumour efficacy of oncolytic viral therapies. However, NK cells are also potent components of the host anti-viral immune response, and therefore have the potential for detrimental anti-viral responses, limiting the spread and persistence of oncolytic viruses. Oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma (HCC), a leading cause of cancer-related death with a high unmet clinical need. In this review, we highlight the role of NK cells in oncolytic virus therapy, their potential for improving treatment options for patients with HCC, and discuss current and potential strategies targeting NK cells in combination with oncolytic viral therapies.

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors.

BACKGROUND: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit. METHODS: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed. RESULTS: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102. CONCLUSION: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.

Activating killer cell immunoglobulin-like receptors: Detection, function and therapeutic use.

Killer cell immunoglobulin-like receptors (KIRs) have a central role in the control of natural killer (NK) cell function. The functions of the activating KIRs, as compared to those of the inhibitory KIR, have been more difficult to define due to difficulties in antibody-mediated identification and their apparent low affinities for HLA class I. Immunogenetic studies have shown associations of activating KIRs with the outcome of autoimmune diseases, pregnancy-associated disorders, infectious diseases and cancers. Activating KIR are thus thought to have important roles in the control of natural killer cell functions and their role in disease. In this review, we discuss current knowledge on activating KIR, their ligands and, their roles in the pathogenesis and potential therapy of human diseases.

Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis.

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 (p < 0.005), and enhanced IFNγ production (p < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups (p < 0.001 vs. healthy controls and p < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a "primed" phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.

A novel antibody combination to identify KIR2DS2high natural killer cells in KIR2DL3/L2/S2 heterozygous donors.

The killer cell immunoglobulin-like receptor (KIR) KIR2DS2 induces natural killer (NK) cell activation upon ligation and in genetic studies is associated with protection against certain cancers and viral infections. One of the difficulties in understanding KIR2DS2 has been that ligands have been hard to define. In part, this is because the high sequence homology between KIR2DS2 and KIR2DL3/KIR2DL2 has made it difficult to make antibodies that specifically detect NK cells expressing KIR2DS2. Using transfected NK cell line (NKL) cells and primary human samples, we report the identification of a novel antibody combination which allows identification of NK cells with relatively high expression of KIR2DS2. This separation is sufficient to examine primary human NK cell activation in response to KIR2DS2 specific ligands.

Hydrogen Peroxide Triggers a Dual Signaling Axis To Selectively Suppress Activated Human T Lymphocyte Migration.

H2O2 is an early danger cue required for innate immune cell recruitment to wounds. To date, little is known about whether H2O2 is required for the migration of human adaptive immune cells to sites of inflammation. However, oxidative stress is known to impair T cell activity, induce actin stiffness, and inhibit cell polarization. In this study, we show that low oxidative concentrations of H2O2 also impede chemokinesis and chemotaxis of previously activated human T cells to CXCL11, but not CXCL10 or CXCL12. We show that this deficiency in migration is due to a reduction in inflammatory chemokine receptor CXCR3 surface expression and cellular activation of lipid phosphatase SHIP-1. We demonstrate that H2O2 acts through an Src kinase to activate a negative regulator of PI3K signaling, SHIP-1 via phosphorylation, providing a molecular mechanism for H2O2-induced chemotaxis deficiency. We hypothesize that although H2O2 serves as an early recruitment trigger for innate immune cells, it appears to operate as an inhibitor of T lymphocyte immune adaptive responses that are not required until later in the repair process.

The Dual Syk/JAK Inhibitor Cerdulatinib Antagonizes B-cell Receptor and Microenvironmental Signaling in Chronic Lymphocytic Leukemia.

Purpose: B-cell receptor (BCR)-associated kinase inhibitors, such as ibrutinib, have revolutionized the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative, and resistance is already emerging in a proportion of patients. IL4, expressed in CLL lymph nodes, can augment BCR signaling and reduce the effectiveness of BCR kinase inhibitors. Therefore, simultaneous targeting of the IL4- and BCR signaling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients.Experimental Design: PBMCs from patients with CLL were treated in vitro with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine, and cell signaling assay were performed and analyzed by flow cytometry, immunoblotting, q-PCR, and ELISA as indicated.Results: At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL4-induced downstream signaling in CLL cells using multiple readouts and prevented anti-IgM- and nurse-like cell (NLC)-mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV-unmutated samples with greater BCR signaling capacity and response to IL4, or samples expressing higher levels of sIgM, CD49d+, or ZAP70+ Cerdulatinib overcame anti-IgM, IL4/CD40L, or NLC-mediated protection by preventing upregulation of MCL-1 and BCL-XL; however, BCL-2 expression was unaffected. Furthermore, in samples treated with IL4/CD40L, cerdulatinib synergized with venetoclax in vitro to induce greater apoptosis than either drug alone.Conclusions: Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. Clin Cancer Res; 23(9); 2313-24. ©2016 AACR.

IL-4 enhances expression and function of surface IgM in CLL cells.

Kinase inhibitors targeting the B-cell receptor (BCR) are now prominent in the treatment of chronic lymphocytic leukemia (CLL). We have focused here on interleukin 4 (IL-4), a cytokine that protects normal and malignant B cells from apoptosis and increases surface immunoglobulin M (sIgM) expression on murine splenic B cells. First, we have demonstrated that IL-4 treatment increased sIgM expression in vitro on peripheral blood B cells obtained from healthy individuals. In CLL, IL-4 target genes are overexpressed in cells purified from the lymph nodes of patients compared with cells derived from matched blood and bone marrow samples. As for normal B cells, IL-4 increased sIgM expression on CLL cells in vitro, especially in samples expressing unmutated V-genes. IL-4-induced sIgM expression was associated with increased receptor signalling activity, measured by anti-IgM-induced calcium mobilization, and with increased expression of CD79B messenger RNA and protein, and the "mature" glycoform of sIgM. Importantly, the ability of the BCR-associated kinase inhibitors idelalisib and ibrutinib, approved for treatment of CLL and other B-cell malignancies, to inhibit anti-IgM-induced signalling was reduced following IL-4 pretreatment in samples from the majority of patients. In contrast to stimulatory effects on sIgM, IL-4 decreased CXCR4 and CXCR5 expression; therefore, CLL cells, particularly within the progressive unmutated V-gene subset, may harness the ability of IL-4 to promote BCR signalling and B-cell retention within lymph nodes. Effects of IL-4 were mediated via JAK3/STAT6 and we propose a potential role for JAK inhibitors in combination with BCR kinase inhibitors for the treatment of CLL.

Pharmacological targeting of PI3K isoforms as a therapeutic strategy in chronic lymphocytic leukaemia.

PI3Kδ inhibitors such as idelalisib are providing improved therapeutic options for the treatment of chronic lymphocytic leukaemia (CLL). However under certain conditions, inhibition of a single PI3K isoform can be compensated by the other PI3K isoforms, therefore PI3K inhibitors which target multiple PI3K isoforms may provide greater efficacy. The development of compounds targeting multiple PI3K isoforms (α, ß, δ, and γ) in CLL cells, in vitro, resulted in sustained inhibition of BCR signalling but with enhanced cytotoxicity and the potential for improve clinical responses. This review summarises the progress of PI3K inhibitor development and describes the rationale and potential for targeting multiple PI3K isoforms.