Downregulation Of NKG2D Ligands Research Articles

KLF4-mediated upregulation of the NKG2D ligand MICA in acute myeloid leukemia: a novel therapeutic target identified by enChIP

The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo.D1rDmzAPriWUyYABRWPo9PVideo

P480: NKG2D-MEDIATED ANTI-TUMOR IMMUNITY CONTRIBUTES TO THE FAVORABLE PROGNOSIS IN APL

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor prognosis. Acute promyelocytic leukemia (APL) is a unique subtype of AML, with PML-RARA fusion caused by t(15;17)(q22;q12) translocation, and is now considered a highly curable disease. Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. NKG2D is an activating receptor on NK cells that recognizes ligands on the surface of tumor cells and plays a crucial role in tumor cell killing. Prior studies have demonstrated that one mechanism of AML immune evasion is downregulation of NKG2D ligands (NKG2DL) or secretion of receptor-blocking soluble ligands. Whereas, reports on NKG2DL expression in APL are still lacking. In this study, we investigate the differences of NKG2DL expression on APL and non-APL cells. Aims: Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. Methods: Bone marrow samples were derived from de novo APL and non-APL AML patients, diagnosed according to French-American-British (FAB) criteria in The First Affiliated Hospital of Xi’an Jiao Tong University. Flow cytometry was performed with the anti-NKG2DL mAbs (MIC-A/B-PE, ULBP-1-APC, ULBP-2/5/6-PerCP). Leukemic blasts were identified using the CD45/SSC gating procedure. Non-APL AML patients were divided into high or low group based on the median expression levels of NKG2DL for survival analysis. Results: Totally 46 patients comprising of 23 APL and 23 non-APL AML were enrolled in this study. There was no significant difference in the baseline characteristics between the two groups. All non‐APL AML cells had low or no NKG2DL surface expression, while most APL cells expressed high NKG2DL levels. In particular, just two APL patients was negative for the NKG2DL but positive for CD34. The median percentage of NKG2DL among non-APL AML cells and APL cells were 1.2% vs 20.3% in MIC-A/B (P<0.01), 1.1% vs 54.8% in ULBP-1 (P=0.017), and 3.8% vs 36.35% in ULBP-2/5/6 (P<0.01). (Fig.1) 23 non-APL AML patients were enrolled for study inclusion; one patient dropped out due to cerebral infarction. The median follow-up time was 17.2 weeks. The high ULBP-2/5/6 group showed a superior overall survival rate compared to the low ULBP-2/5/6 group (P < 0.01), but there was no significant difference between high and low MIC-A/B groups (P = 0.31) or between the high and low ULBP-1 groups (P = 0.52). Image:Summary/Conclusion: NKG2DL was decreased significantly on the surface of non-APL AML cells compared with APL cells. These results may indicate that NKG2D-mediated anti-tumor immunity contributes to the favorable prognosis in APL, while immune escape is an underlying mechanism for unfavorable prognosis in non-APL AML. NKG2DL may be used as a potential biomarker to predict prognosis of patients with non-APL AML, and upregulation of its expression could improve the clinical efficacy. Further studies with larger sample size and longer follow-up are required to confirm our conclusion.

Bispecific antibody-mediated redirection of NKG2D-CAR natural killer cells facilitates dual targeting and enhances antitumor activity

BackgroundNatural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as...

WITHDRAWN: Epithelial-mesenchymal transition may be involved in the immune evasion of circulating gastric tumor cells via downregulation of NKG2D ligands

// Baoguang Hu 1, * , Xiaokun Tian 1, 2, * , Yanbin Li 1 , Yuming Li 1 , Zhaodong Han 3 , Jiajia An 3 and Lingqun Kong 4 1 Department of Gastrointestinal Surgery, The Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, China 2 Department of Burn and Plastic Surgery, The Sixth People’s Hospital of Zibo, Zibo, Shandong, China 3 Department of Clinical Laboratory, The Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, China 4 Department of Hepatobiliary Surgery, The Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, China * These authors have contributed equally to this work Correspondence to: Yu-ming Li, email: PIliym@126.com Jiajia An, email: jiajiabest_007@163.com Keywords: gastric cancer; circulating tumor cells; immune evasion; epithelial-mesenchymal transition; transforming growth factor-beta 1 Received: July 11, 2017 Accepted: November 17, 2017 Published: January 02, 2018 ABSTRACT Increasing numbers of studies have demonstrated that circulating tumor cells (CTCs) undergo a phenotypic change termed epithelial-mesenchymal transition (EMT), and researchers have proposed that EMT might provide CTCs with increased potential to survive in the different microenvironments encountered during metastasis through various ways, such as by enhancing metastatic competence, and increasing cell survival and early colonization. However, the exact role of EMT in CTCs remains unclear. In this study, we identified CTCs in the peripheral blood of 41 patients with gastric cancer using Cyttel-CTC and im-FISH (immune-fluorescence in situ hybridization) methods, and tested the expression of EMT markers and NK-cell receptor D (NKG2D) ligands on CTCs. Moreover, we investigated the relationship between the expression of EMT markers and NKG2D ligands on CTCs and gastric cancer cell lines. The results showed that the CTCs in the peripheral blood of gastric cancer patients exhibited three EMT marker subtypes, and that the expression of NKG2D ligands was significantly lower on mesenchymal phenotypic CTCs (M + CTCs) than on epithelial phenotypic CTCs (E + CTCs). EMT induced by TGF-β in vitro produced a similar phenomenon, and we therefore proposed that EMT might be involved in the immune evasion of CTCs from NK cells by altering the expression of NKG2D ligands. In conclusion, our study indicated that EMT might play a key role in the immune invasion of gastric CTCs by regulating the expression of NKG2D ligands on the tumor cells. These findings could provide potential strategies for targeting the immune evasion capacity of CTCs.

Abstract 5591: Mek/erk inhibition enhances anti-pd-l1 effects on the susceptibility to NK cytotoxicity of cisplatin-resistant non-small cell lung cancer

Abstract Background: Immunotherapy is often used after chemotherapy failure or in combination with chemotherapy. Initial chemotherapy may yield clinical benefit, however; after continuous chemotherapy treatments, chemoresistance invariably develops. Immunotherapy has shown effects in a fraction of patients, but not all patients with chemoresistant non-small cell lung cancer (NSCLC) will respond. We investigated a new mechanism to enhance natural killer (NK) cell cytotoxicity against cisplatin-resistant NSCLC and propose to inhibit MEK/Erk pathway to enhance anti-PD-L1 effects on the susceptibility to NK cytotoxicity of cisplatin-resistant NSCLC. Methods: NK cell mediated cytotoxicity was conducted using two NK cell sources: NK92 cell line and primary NK cells. LDH release-based NK cytotoxicity test and the colony formation assay were applied to monitor NK cytotoxicity. NK cell cytotoxicities to parental vs. cisplatin-resistant A549 and H157 sublines (A549CisR and H157CisR) were compared. We investigated signaling molecules/pathways that are responsible for the constitutively up-regulated expression of PD-L1 and down-regulated NKG2D ligands in cisplatin-resistant cells using qPCR and Western blot analyses and inhibition studies using inhibitors of each signaling pathway were conducted in parallel. Results: We found cisplatin-resistant NSCLC cells were resistant to NK cell cytotoxicity, due to the up-regulation of PD-L1 and down-regulation of NKG2D ligands in A549CisR and H157CisR cells compared to parental cells. In addition, we found that the expression of PD-1 in NK cells was induced after co-culture with cisplatin-resistant cells, but not with parental cells. Susceptibility of cisplatin-resistant cells to NK cell cytotoxicity increased when the PD-L1/PD-1 interaction was inhibited by Abs of PD-1 or PD-L1. In inhibitor studies, we found that the inhibition of MEK/Erk signaling effectively reduced the PD-L1 level while recovered the NKG2D ligands levels in cisplatin-resistant cells. Subsequently, adding the MEK/Erk inhibitor enhanced the PD-L1 Ab effect in increasing the susceptibility of A549CisR and H157CisR cells to NK cell cytotoxic action. Conclusion and Impacts: Our data suggest that the inhibition of MEK/Erk pathway may enhance the anti PD-L1/PD-1 therapy efficacy in increasing the susceptibility of cisplatin-resistant cells to NK cell cytotoxicity via reducing the constitutively expressed PD-L1 levels, while simultaneously recovering NKG2D ligand levels in these cells. This strategy of dual effects has the therapeutic potential for molecular targeted drug development. The immune escape of cisplatin-resistant cells is an emerging task to overcome in lung cancer therapeutics, thus positive outcome of our studies may result in clinical significance. Citation Format: MingJing Shen, LiJun Xu, Ying Tsai, Peter C. Keng, Soo Ok Lee, Yuhchyau Chen. Mek/erk inhibition enhances anti-pd-l1 effects on the susceptibility to NK cytotoxicity of cisplatin-resistant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5591. doi:10.1158/1538-7445.AM2017-5591

Abstract 3235: IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands

Abstract Background: Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Gain of function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. Methods: We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza- 2’deoxycytodine (Decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. Results: All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell- mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK-mediated lysis of IDH mutant cells in an NKG2D- dependent manner. Conclusions: IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell- mediated immune surveillance in patients with IDH mutated diffuse gliomas. Citation Format: Aparna Rao, Xiaoran Zhang, Christopher Deibert, Paola Sette, Tim Chan, Paola Grandi, Nduka Amankulor. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3235.

γδ T cells as a potential tool in colon cancer immunotherapy.

γδ T cells are capable of recognizing tumor cells and exert potent cellular cytotoxicity against a large range of tumors, including colon cancer. However, tumors utilize numerous strategies to escape recognition or killing by patrolling γδ T cells, such a downregulation of NKG2D ligands, MICA/B and ULBPs. Therefore, the combined upregulation of T-cell receptorand NKG2D ligands on tumor cells and induction of NKG2D expression on γδ T cells may greatly enhance tumor killing and unlock the functions of γδ T cells. Here, we briefly review current data on the mechanisms of γδ T-cell recognition and killing of colon cancer cells and propose that γδ T cells may represent a promising target for the design of novel and highly innovative immunotherapy in patients with colon cancer.

The role of NKG2D signaling in inhibition of cytotoxic T-lymphocyte lysis by the Murine cytomegalovirus immunoevasin m152/gp40.

Three proteins encoded by murine cytomegalovirus (MCMV) -- gp34, encoded by m04 (m04/gp34), gp48, encoded by m06 (m06/gp48), and gp40, encoded by m152 (m152/gp40) -- act together to powerfully impact the ability of primed cytotoxic CD8 T lymphocytes (CTL) to kill virus-infected cells. Of these three, the impact of m152/gp40 on CTL lysis appears greater than would be expected based on its impact on cell surface major histocompatibility complex (MHC) class I. In addition to MHC class I, m152/gp40 also downregulates the RAE-1 family of NKG2D ligands, which can provide costimulation for CD8 T cells. We hypothesized that m152/gp40 may impact CTL lysis so profoundly because it inhibits both antigen presentation and NKG2D-mediated costimulation. We therefore tested the extent to which m152/gp40's ability to inhibit CTL lysis of MCMV-infected cells could be accounted for by its inhibition of NKG2D signaling. As was predictable from the results reported in the literature, NKG2D ligands were not detected by NKG2D tetramer staining of cells infected with wild-type MCMV, whereas those infected with MCMV lacking m152/gp40 displayed measurable levels of the NKG2D ligand. To determine whether NKG2D signaling contributed to the ability of CTL to lyse these cells, we used a blocking anti-NKG2D antibody. Blocking NKG2D signaling did affect the killing of MCMV-infected cells for some epitopes. However, for all epitopes, the impact of m152/gp40 on CTL lysis was much greater than the impact of inhibition of NKG2D signaling. We conclude that the downregulation of NKG2D ligands by MCMV makes only a small contribution to the impact of m152/gp40 on CTL lysis and only for a small subset of CTL.

Human cytomegalovirus glycoprotein UL16 causes intracellular sequestration of NKG2D ligands, protecting against natural killer cell cytotoxicity.

The activating receptor, NKG2D, is expressed on a variety of immune effector cells and recognizes divergent families of major histocompatibility complex (MHC) class I–related ligands, including the MIC and ULBP proteins. Infection, stress, or transformation can induce NKG2D ligand expression, resulting in effector cell activation and killing of the ligand-expressing target cell. The human cytomegalovirus (HCMV) membrane glycoprotein, UL16, binds to three of the five known ligands for human NKG2D. UL16 is retained in the endoplasmic reticulum and cis-Golgi apparatus of cells and causes MICB to be similarly retained and stabilized within cells. Coexpression of UL16 markedly reduces cell surface levels of MICB, ULBP1, and ULBP2, and decreases susceptibility to natural killer cell–mediated cytotoxicity. Domain swapping experiments demonstrate that the transmembrane and cytoplasmic domains of UL16 are important for intracellular retention of UL16, whereas the ectodomain of UL16 participates in down-regulation of NKG2D ligands. The intracellular sequestration of NKG2D ligands by UL16 represents a novel HCMV immune evasion mechanism to add to the well-documented viral strategies directed against antigen presentation by classical MHC molecules.