Diacylglycerol (DG) kinase (DGK), which phosphorylates DG to generate phosphatidic acid (PA), consists of ten isozymes (α-к). Recently, we identified a novel small molecule inhibitor, CU-3, that selectively inhibits the activity of the α isozyme. In addition, we newly obtained Compound A, which selectively and strongly inhibits type I DGKs (α, β, and γ). In the present study, we demonstrated that both CU-3 and Compound A induced apoptosis (caspase 3/7 activity and DNA fragmentation) and viability reduction of AKI melanoma cells. Liquid chromatography-mass spectrometry revealed that the production of 32:0-and 34:0-PA species was commonly attenuated by CU-3 and Compound A, suggesting that lower levels of these PA molecular species are involved in the apoptosis induction and viability reduction of AKI cells. We determined the effects of the DGKα inhibitors on several other cancer cell lines derived from refractory cancers. In addition to melanoma, the DGKα inhibitors enhanced caspase 3/7 activity and reduced the viability of hepatocellular carcinoma, glioblastoma, and pancreatic cancer cells, but not breast adenocarcinoma cells.Interestingly, Western blot analysis indicated that the DGKα expression levels were positively correlated with the sensitivity to the DGK inhibitors. Because both CU-3 and Compound A induced interleukin-2 production by T cells, it is believed that these two compounds can enhance cancer immunity. Taken together, our results suggest that DGKα inhibitors are promising anticancer drugs. K E Y W O R D S apoptosis, cancer, diacylglycerol kinase (DGK), inhibitor, melanoma, phosphatidic acid (PA)

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.

Introduction Diacyglycerol kinase δ isozyme (DGKδ) plays critical roles in lipid signaling by phosphorylating diacylglycerol (DG) into phosphatidic acid (PA). DGKδ regulates a wide variety of physiological and pathological events, such as type II diabetes and obsessive compulsive disorder. Because DGK is one of the components of phosphatidylinositol (PI) turnover, it is thought that DGKδ also utilizes mainly 18:0/20:4‐DG (X:Y; the total number of carbon atoms: the total number of double bonds) derived from PI turnover. Interestingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐containing DG molecular species, but not arachidonic acid (20:4)‐containing DG species, in response to high glucose stimulation. However, it is still unclear what kind of DG‐generating enzyme provides palmitic acid‐containing DG species. Sphingomyelin synthase (SMS) 1, SMS2 and SMS‐related protein (SMSr) are DG‐generating enzymes utilizing phosphatidylcholine/phosphatidylethanolamine and ceramide. SMS1 and SMSr contain a sterile α motif domain (SAM), which is a protein‐protein interaction module, at their N‐termini. DGKδ also possesses SAM at its C‐terminus, and DGKδ‐SAM is highly homologous to SMSr‐SAM. Therefore, we hypothesised that DGKδ interacts with SMSr through their SAMs. In the present study, we investigated the interaction between DGKδ‐SAM and SMSr‐SAM. Results We first examined whether DGKδ‐SAM interacts with SMSr‐SAM by immunoprecipitation analysis. We found that SMSr‐SAM, but not SMS1‐SAM, was co‐immunoprecipitated with DGKδ‐SAM. Full‐length DGKδ (DGKδ‐FL) was also co‐immunoprecipitated with SMSr‐FL more strongly than with SMS1‐FL and with SMS2‐FL. To examine whether SMSr‐SAM contribute to the interaction, we performed immunoprecipitation analysis using SMSr‐FL and a SAM‐deletion mutant (SMSr‐DSAM). DGKδ‐FL was strongly co‐immunoprecipitated with SMSr, whereas SMSr‐DSAM only weakly interacted with DGKδ‐FL. Immunostaining analysis demonstrated that DGKδ‐FL co‐localized partly with SMSr‐FL in COS‐7 cells overexpressing these proteins. These results strongly suggest that DGKδ interacts with SMSr through their SAMs. Conclusion In summary, the present study for the first time showed that DGKδ interacted with SMSr mainly through their SAMs. It is possible that SMSr is one of the candidates of up stream DG‐providing enzymes of DGKδ, which composes a new pathway independent of PI turnover. Support or Funding Information This work was supported by JSPS KAKENHI (Grant Number JP18J20003) and supported in part by Venture Business Laboratory in Chiba University (Nanohana Competition 2018 Award). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

These results suggest that the chronic oral administration of myristic acid improves hyperglycaemia by decreasing insulin-responsive glucose levels and reducing body weight, and that the fatty acid accounts for the diabetes protective properties of high-fat dairy products. Myristic acid is a potential candidate for the prevention and treatment of type 2 diabetes mellitus and its related diseases.

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