Nectin2 is a member of immunoglobulin-like cell adhesion molecules and plays a prominent role in the establishment of adherens and tight junctions. It is also upregulated on the surface of tumor and virus-infected cells where it functions as a ligand for the activating receptor CD226, thus contributing to cytotoxic lymphocyte-mediated recognition and killing of damaged cells. Little is currently known about the regulation of Nectin2 expression and, in particular, whether posttranscriptional and posttranslational mechanisms are involved. Here, we analyzed Nectin2 expression on a panel of human tumor cell lines and primary cultures and we found that Nectin2 is mainly expressed in cytoplasmic pools. Moreover, we demonstrated that ubiquitination of Nectin2 promotes its degradation and is responsible for protein intracellular retention. Indeed, inhibition of the ubiquitin pathway results in increased Nectin2 surface expression and enhances tumor cell susceptibility to NK cell cytotoxicity. Our results demonstrate a previously unknown mechanism of Nectin2 regulation revealing that the ubiquitin pathway represents a potential target of intervention in order to increase susceptibility to NK cell-mediated lysis.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells.
Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138+ MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays.
Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA.
These findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.

Not all natural killer (NK) cells are equally cytotoxic against leukemia because of differences in receptor gene content and surface expression. We correlated NK cell genotype and phenotype at diagnosis of childhood acute lymphoblastic leukemia (ALL) with minimal residual disease (MRD) after induction chemotherapy.The NK cells and leukemia blasts of 244 patients were analyzed at diagnosis by killer-cell immunoglobulin-like receptor (KIR) typing and immunophenotyping. The results were correlated statistically with postinduction MRD status.The odds of being MRD positive in patients with KIR telomeric (Tel)-A/B genotype were 2.85 times the odds in those with Tel-A/A genotype (P = 0.035). MRD-positive patients were more likely to have KIR2DL5A (P = 0.006) and expressed less activating receptor NKp46 and FASL on their NK cells (P = 0.0074 and P = 0.029, respectively). The odds of being MRD positive increased by 2.01-fold for every percentage increase in NK cells expressing KIR2DL1 in the presence of HLA-C2 ligand (P = 0.034). The quantity of granzyme B inhibitor PI-9 in the leukemia blasts was greater in patients who were MRD positive (P = 0.038). Collectively, five NK cell-related factors (Tel-B-associated KIR2DL5A, NKp46, FASL, granzyme B, and PI-9) are strongly associated with MRD positivity at the end of induction with 100% sensitivity and 80% specificity.Our data support the hypothesis that NK cells with a strong effector phenotype in the setting of decreased leukemia resistance are associated with better leukemia control.©2014 American Association for Cancer Research.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA.
© 2014 John Wiley & Sons Ltd.