Cancer metastasis is the leading cause of death in patients with any type of solid cancer. To develop effective cancer treatments, it is essential to understand the molecular mechanisms underlying cancer metastasis. Previously we established peritoneal metastasis cell models derived from human scirrhous gastric cancer patients. In this article, we focus on the CELSR1 gene, which is involved in Wnt signaling but whose association with peritoneal metastasis is still unclear. We unveiled gene alterations and the prognostic relevance of the CELSR1 gene in cancer patients by analyzing public resources for cancer genomic and patient cohorts. RT-qPCR and immunoblot analyses revealed that CELSR1 expression was significantly elevated in our cell lines, which had high peritoneal metastatic property, compared with their parental cell lines, which had lower peritoneal metastatic property. Some of the gene alterations in the coding region of CELSR1 were observed in our metastatic cell lines, but they were not associated with the metastatic property or with patient prognosis. Knockdown of CELSR1 via the shRNA technique significantly decreased migration and invasion in the cell lines having high peritoneal metastatic property, whereas the knockdown did not significantly affect proliferation. These results show that CELSR1 plays an important role in peritoneal metastasis and that CELSR1 is a novel peritoneal metastasis-associated gene. The results also suggest that CELSR1 is a proper molecular target for therapy against peritoneal metastasis of scirrhous gastric cancers.

T-cell receptor-engineered T-cell therapies have achieved promising response rates against synovial sarcoma in clinical trials, but their applicability is limited owing to the HLA matching requirement. Chimeric antigen receptor (CAR) can redirect primary T cells to tumor-associated antigens without requiring HLA matching. However, various obstacles, including the paucity of targetable antigens, must be addressed for synovial sarcoma. Ligands for natural killer (NK) cell-activating receptors are highly expressed by tumor cells. The surface expression of ligands for NK cell-activating receptors in synovial sarcoma cell lines was analyzed. We analyzed RNA sequencing data deposited in a public database to evaluate NKp44-ligand expression. Primary T cells retrovirally transduced with CAR targeting NKp44 ligands were evaluated for their functions in synovial sarcoma cells. Alterations induced by various stimuli, including a histone deacetylase inhibitor, a hypomethylating agent, inflammatory cytokines, and ionizing radiation, in the expression levels of NKp44 ligands were investigated. : Ligands for NKp44 and NKp30 were expressed in all cell lines. NKG2D ligands were barely expressed in a single cell line. None of the cell lines expressed NKp46 ligand. Primary synovial sarcoma cells expressed the mRNA of the truncated isoform of MLL5, a known cellular ligand for NKp44. NKp44-based CAR T cells specifically recognize synovial sarcoma cells, secrete interferon-γ, and exert suppressive effects on tumor cell growth. No stimulus altered the expression of NKp44 ligands. NKp44-based CAR T cells can redirect primary human T cells to synovial sarcoma cells. CAR-based cell therapies may be an option for treating synovial sarcomas.

Abstract 1901 Majority of AML blasts express CD38, while human hematopoietic stem cells express CD34 but not CD38. Accordingly, we focused on CD38 for a therapeutic target and developed a cellular immunotherapy by using T cells bearing anti-CD38-chimeric antigen receptor (CAR). We recently reported T cells with anti-CD38-CAR efficiently eliminated B-cell lymphoma cells expressing CD38 in vitro and in vivo. However, intensity of CD38 in lymphoma cells is much higher than that in AML cells. Additionally, leukemic stem cells(LSCs) phenotypically express CD34⁺CD38⁻. Thus, to fully employ anti-CD38-CAR against AML blasts including LSCs, intensity of CD38 expression need to be raised for the clinical application. All-trans retinoic acid (ATRA) is widely used for treatment of patients with acute promyelocytic leukemia (APL). Interestingly, it has been reported that ATRA enhances CD38 expression on the surface of AML cells. In this study, we investigated whether human peripheral T cells retrovirally transduced with anti-CD38-CAR kills AML cells and furthermore, whether the cytotoxicity is enhanced in the presence of ATRA. First of all, we confirmed that T cells bearing anti-CD38-CAR expressed GFP as well as the anti-goat mouse IgG-PerCP, whose expression is consistent with the expression of anti-CD38-CAR on the T cells. Next, we evaluated the cytotoxic effect of CD38-specific T cells against two AML cell lines (THP-1 and CMK), which highly express CD38 (>99%). Lactate dehydrogenase (LDH) releasing assay and flow cytometry were performed for cytotoxicity. We co-incubated THP-1 or CMK cells with T cells transduced at a variety of effector (E): target (T) ratios for 3 consecutive days in vitro. Interestingly, these assays showed that T cells bearing anti-CD38-CAR were cytotoxic against THP-1 and CMK cells in dose- and time-dependent manners. However, in the cases of KG1a, U937 and HL60 cells which partially express CD38, killing effect was restricted to CD38⁺ cells by T cells bearing anti-CD38-CAR, and CD38⁻ AML cells remained alive. These results suggested that augmentation of CD38 expression is essential for the sufficient cytotoxicity against AML cells not expressing CD38 by T cells bearing anti-CD38-CAR. We, then, investigated whether ATRA augmented CD38 expression and resultantly enhanced the cytotoxicity against AML cell lines. Interestingly, ATRA augmented CD38 expression in HL60 cells as well as even in KG1a and U937. However, ATRA by itself exerted no effect on cytotoxicity or proliferating activity in KG1, U937 and even in HL60 cells. These results showed that ATRA contributed to enhancement of CD38 expression, but not to cytotoxicity and proliferation on AML cell lines. Next, we attempted to examine cytotoxic effect of T cells with anti-CD38-CAR on KG1, U937 or HL60 cells following ATRA treatment. AML cell lines were co-cultured with the effector T cells transduced for 3 days in the presence of ATRA. The killing effect of T cells bearing anti-CD38-CAR against AML cell lines was limited in the absence of ATRA. Intriguingly, T cells with anti-CD38-CAR exerted enhanced cytotoxic effect on AML cells in the presence of ATRA. Next, we applied our settings above to AML cells freshly isolated from AML patients. Firstly, we confirmed that CD38 expression was enhanced by ATRA in AML cells from patients as observed in AML cell lines. Furthermore, 3-day incubation of patients' AML cells with T cells bearing anti-CD38-CAR abrogated AML cells in the presence of ATRA. These results indicated that T cells expressing anti-CD38-CAR efficiently eliminated AML cells from patients as well as AML cell line cells through the enhancement of CD38 expression by ATRA. Here, we propose that pre-treatment of ATRA in patient with AML enhances CD38 expression on the leukemic cell surface, which augments the cytotoxic effect of T cells bearing CAR to eradicate leukemic cells including LSCs.

Programmed death 1 ligand (PD-L1), an important immune checkpoint molecule, is mainly expressed on cancer cells and has been shown to exert an immunosuppressive effect on T-cell function by binding to programmed cell death 1 (PD-1) expressed on T-cells. Recently, immune checkpoint inhibitors using antibody drugs such as nivolumab and atezolizumab have attracted attention. However, clinical challenges, including limitations to the scope of their application, are yet to be addressed. In this study, we developed a novel immune checkpoint inhibitor that targets PD-L1 using lipid-siRNA conjugates (lipid-siPDL1s). The inhibitory effect of lipid-siPDL1s on PD-L1 expression was evaluated and found to strongly suppress mRNA expression. Notably, lipid-siPDL1s exerted a significantly stronger effect than unmodified siPDL1. Interestingly, lipid-siPDL1s strongly inhibited PD-L1 expression despite cancer cell stimulation by interferon-gamma, which induced the overexpression of PD-L1 genes. These results strongly suggest that lipid-siPDL1s could be used as novel immune checkpoint inhibitors.

Aggressive periodontitis causes rapid destruction of periodontal tissue. It occurs at a young age with familial clustering. We report on the first time on molecular and cellular basis of a Mendelian form of autosomal dominant aggressive periodontitis. Monoallelic mutations in the monocyte to macrophage differentiation-associated 2 (MMD2) gene, encoding MMD2, in two Japanese families with autosomal dominant aggressive periodontitis are identified. Mutations, c.347 C>T (p.A116V) and c.377 G>C (p.R126P) in MMD2, disturbed fMLP-induced activation of Ras/ERK signaling. Additionally, abnormalities in the proteins of Golgi apparatus, a crucial contributor to innate immune signaling pathways, were identified in patients’ neutrophils. The knock-in and knockout mice exhibited alveolar bone loss by ligature-induced periodontitis, along with impaired fMLP-induced chemotaxis, as found in the patients with MMD2 mutation. Our studies revealed that monoallelic mutations in MMD2 underlie the impairment of neutrophil chemotaxis, which leads to the development of autosomal dominant aggressive periodontitis.