Abstract
The diacylglycerol kinases (DGKs) are a family of lipid kinases whose primary function is the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). There has been mounting evidence indicating that DGK enzymes are implicated in other physiologic processes, ranging from immune cell regulation to cancer progression. DGKα in particular is a known promoter of T-cell anergy, and has been demonstrated as a promising therapeutic target in multiple cancers including glioblastoma (GBM) and melanoma. Prior to these following studies, the only significant phenotype observed in DGKα knockout (KO) mice has been enhanced T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to activating stimuli. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also had increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains.
Targeting DGKα in order to stimulate T-cell function or to combat its tumorigenic effects in cancer is feasible due to the existence of several small-molecule inhibitors. R59949 and R59022 are tool compounds which limit the DGKα conversion of DAG to PA. The compound ritanserin was originally implicated clinically as a serotonin receptor antagonist used for the treatment of schizophrenia and has been used in clinical trial settings with no demonstrated adverse effects. Likely due to ritanserin’s structural similarity to R59949 and R59022, it is also capable of inhibiting DGKα, with the added benefits of safety and being clinically actionable. In the following studies, we have identified several additional and potentially clinically relevant small molecule compounds from a library of ritanserin analogs which are more potent and specific for DGKα than those identified previously. Effective targeting of DGKα has promising therapeutic implications, and our newly identified immune regulatory role of DGKα is further support for the appeal of developing these compounds. Taken together, these studies present a novel immune-regulatory function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings, and the identification of inhibitory compounds to translate these findings therapeutically.
