Diacylglycerol Kinase Alpha Research Articles

Diacylglycerol kinase alpha is a proliferation marker of intrahepatic cholangiocarcinoma associated with the prognosis.

Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. Immunohistochemistry (IHC) with anti-DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (-, 1+, 2+, 3+) and divided into two groups (DGKα- and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki-67 expression was evaluated as a cell proliferation marker. The number of Ki-67-positive cells was counted, and the relationship with DGKα expression was examined. DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα- group (-: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence-free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki-67-positive cells increased in association with the staining levels of DGKα. Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.

Ritanserin suppresses acute myeloid leukemia by inhibiting DGKα to downregulate phospholipase D and the Jak-Stat/MAPK pathway

Refractory or relapsed (R/R) AML is the most challenging form of AML to treat. Due to frequent genetic mutations, therapy alternatives are limited. Here, we identified the role of ritanserin and its target DGKα in AML. Several AML cell lines and primary patient cells were treated with ritanserin and subjected to cell proliferation, apoptosis and gene analyses with CCK-8 assay, Annexin V/PI assay and Western blotting, respectively. We also evaluated the function of the ritanserin target diacylglycerol kinase alpha (DGKα) in AML by bioinformatics. In vitro experiments have revealed that ritanserin inhibits AML progression in a dose- and time-dependent manner, and it shows an anti-AML effect in xenograft mouse models. We further demonstrated that the expression of DGKα was elevated in AML and correlated with poor survival. Mechanistically, ritanserin negatively regulates SphK1 expression through PLD signaling, also inhibiting the Jak-Stat and MAPK signaling pathways via DGKα. These findings suggest that DGKα may be an available therapeutic target and provide effective preclinical evidence of ritanserin as a promising treatment for AML.

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.

Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.

Vitamin E functions by association with a novel binding site on the 67 kDa laminin receptor activating diacylglycerol kinase

It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.

Ca2+ -induced structural changes and intramolecular interactions in N-terminal region of diacylglycerol kinase alpha.

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+ -triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα-EF induces conformational changes from a protease-susceptible "open" conformation in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca2+ -binding domain, but the Ca2+ -induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like structure in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+ , and this interaction is modified in the presence of Ca2+ . Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca2+ , which is essential for the activation of the enzyme.

DGKA interacts with SRC/FAK to promote the metastasis of non-small cell lung cancer

Metastasis is responsible for the high mortality rate of lung cancer, but its underlying molecular mechanisms are poorly understood. Here, we demonstrated that the expression of diacylglycerol kinase alpha (DGKA) was elevated in the metastatic lesions of non-small cell lung cancer (NSCLC) and correlated with poor survival. Mechanistic studies revealed a direct physical interaction as well as a mutual regulation among DGKA, proto-oncogene tyrosine-protein kinase Src (SRC), and focal adhesion kinase 1 (FAK) proteins. The C-terminal domain of DGKA was responsible for the SRC SH3 domain binding, while the catalytic domain of DGKA interacted with the FREM domain of FAK. DGKA phosphorylated the SRC protein at Tyr416 and the FAK protein at Tyr397 to form and activate the DGKA/SRC/FAK complex, thus initiating the downstream WNT/β-catenin and VEGF signaling pathways, promoting epithelial-mesenchymal transition (EMT) and angiogenesis, and resulting in the metastasis of NSCLC. DGKA knockdown inhibited the invasive phenotype of NSCLC cells in vitro. Pharmacologic ablation of DGKA inhibited the metastasis of NSCLC cells in vivo, and this was reversed by the overexpression of DGKA. These results suggested that DGKA was a potential prognostic biomarker as well as a promising therapeutic target for NSCLC, especially when there was lymphatic or distant metastasis.

Combination therapy for hepatocellular carcinoma with diacylglycerol kinase alpha inhibition and anti-programmed cell death-1 ligand blockade.

Activation of diacylglycerol kinase alpha (DGKα) augments proliferation and suppresses apoptosis of cancer cells and induces T lymphocyte anergy. We investigated the dual effects of DGKα inhibition on tumorigenesis and anti-tumor immunity with the aim of establishing a novel therapeutic strategy for cancer. We examined the effects of a DGKα inhibitor (DGKAI) on liver cancer cell proliferation and cytokine production by immune cells in vitro and on tumorigenesis and host immunity in a hepatocellular carcinoma (HCC) mouse model. Oral DGKAI significantly suppressed tumor growth and prolonged survival in model mice. Tumor infiltration of T cells and dendritic cells was also enhanced in mice treated with DGKAI, and the production of cytokines and cytotoxic molecules by CD4+ and CD8+ T cells was increased. Depletion of CD8+ T cells reduced the effect of DGKAI. Furthermore, interferon-γ stimulation augmented the expression of programmed cell death-1 ligand (PD-L1) on cancer cells, and DGKAI plus an anti-PD-L1 antibody strongly suppressed the tumor growth. These results suggest that DGKα inhibition may be a promising new treatment strategy for HCC.

Blood Transcriptome Profiling Reveals Diet-Induced Changes in Metabolic Pathway Gene Expression

ObjectivesThis study explored the effects of diet-induced (almonds vs. crackers) changes on blood transcriptome profiles of young adults. MethodsYoung adults (age: 18–22 years) were randomly assigned to consume either almonds (2 oz./d, n = 13) or an isocaloric control snack of graham crackers (325 kcal/d, n = 10) daily for 4 weeks. Blood samples were collected at baseline and 4 weeks after intervention. Total leukocyte RNA was extracted and sequenced. Gene expression profiling was carried out using a 3′ Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit for multiplexed sequencing. Data were preprocessed, STAR aligned, and count tables generated using the QuantSeq FWD-UMI pipeline. Differential expression (DE) analysis was conducted on the time x diet model using the limma-voom packages in R. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the DE p-values was conducted using the Wilcoxon rank-sum test in R. P-values were adjusted for false discovery rate correction (FDR). ResultsOut of 13,018 filtered genes, 69 were differentially expressed (FDR < 0.1). Glutamic-oxaloacetic transaminase 2 (GOT2), diacylglycerol kinase alpha (DGKA), and glutaryl-CoA dehydrogenase (GCDH) genes were upregulated with almond consumption. GOT2 plays a role in amino acid metabolism and the urea and tricarboxylic acid cycles, DGKA is involved in lipid metabolism, and GCDH is involved in lysine degradation and tryptophan metabolism. Cracker consumption resulted in greater upregulation of TGF-beta activated kinase 1 gene (MAP3K7) binding protein 3 (TAB3) gene which is involved in the NF-kappaB signal transduction pathway and a greater downregulation in the immunoglobulin superfamily member 8 (IGSF8) gene. Enrichment analyses indicate gene annotations to 341 KEGG pathways. Thermogeneic and ribosomal pathways were significantly enriched (FDR < 0.1). Pathways related to oxidative phosphorylation, sphingolipid signaling, and tryptophan, propanoate, and starch and sucrose metabolism were also differentially enriched (P < 0.05, FDR < 0.3). ConclusionsThese findings indicate that diet (almond vs cracker) potentially alters metabolism through changes in gene transcription. The implications of these findings and associations with health and disease outcomes need to be investigated further. Funding SourcesNIH

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Simple SummaryTo reduce long-term fibrosis risk after radiotherapy, we demonstrated with different experimental approaches that modulation of the epigenetic pattern at the DGKA enhancer can attenuate pro-fibrotic reactions in human fibroblasts. We used (epi)genomic editing of the DGKA enhancer and administration of various epigenetic drugs and were able to modulate radiation-induced expression of DGKA and pro-fibrotic collagens. Based on our results, clinical application of bromodomain inhibitors will open promising ways to epigenetically modulate DGKA expression and might provide novel therapeutic options to prevent or even reverse radiotherapy-induced fibrotic reactions.Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

DGKA Mediates Resistance to PD-1 Blockade.

Immunologic checkpoint blockade has been proven effective in a variety of malignancies. However, high rates of resistance have substantially hindered its clinical use. Understanding the underlying mechanisms may lead to new strategies for improving therapeutic efficacy. Although a number of signaling pathways have been shown to be associated with tumor cell-mediated resistance to immunotherapy, T cell-intrinsic resistant mechanisms remain elusive. Here, we demonstrated that diacylglycerol kinase alpha (Dgka) mediated T-cell dysfunction during anti-PD-1 therapy by exacerbating the exhaustion of reinvigorated tumor-specific T cells. Pharmacologic ablation of Dgka postponed T-cell exhaustion and delayed development of resistance to PD-1 blockade. Dgka inhibition also enhanced the efficacy of anti-PD-1 therapy. We further found that the expression of DGKA in cancer cells promoted tumor growth via the AKT signaling pathway, suggesting that DGKA might be a target in tumor cells as well. Together, these findings unveiled a molecular pathway mediating resistance to PD-1 blockade and provide a potential therapeutic strategy with combination immunotherapy.

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.

Abstract 4985: PD-L1 and diacylglycerol kinase alpha (DGKA) expression in melanoma, nsclc and bladder cancers

Abstract Diacylglycerol kinase alpha (DGKA) functions as lipid kinase to phosphorylate the lipid diacylglycerol (DAG). DGKA has been characterized to negative regulate the Ras-MAPK pathway in T lymphocytes. A 2018 study using crispr knockouts of DGKA have shown the anti tumor activity of T-cells continue to increase in the presence of PD-L1 positive tumor cells. Little is known about the expression patterns of DGKA in human cancer. In our previous study, we explore the tumor micro environment with a number of immune cell markers such as CD3, CD8A, CD20, CD68, TIM3 and LAG3 with PD-L1 expression in melanoma, NSCLC, and bladder cancer and found a few unique signatures for these cancers. In this study, we evaluate expression pattern of DGKA in various cancers by immunohistochemistry to elucidate the signature profile of DGKA expression with PD-L1 and other markers in the PD-L1 tumor micro environment. Citation Format: Rachel M. Gonzalez, Tianli Qu, Xiaomin Hu, Qi Ren, Qiankun Zhang, Zhaoying Guo, Young-Hwan Kim, Yiran Wang, Xuan Liu, Wei Fu. PD-L1 and diacylglycerol kinase alpha (DGKA) expression in melanoma, nsclc and bladder cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4985.

Abstract 4084: A DGKA-cJUN-WEE1 signaling axis provides platinum resistance in ovarian cancer

Abstract Although platinum compounds are the first-line treatment for ovarian cancer, the majority of patients relapse and develop resistance to treatment. However, the mechanism underlying resistance is unclear. Here, we report a unique platinum resistance mechanism that is mediated by a lipid messenger, phosphatidic acid (PA), in ovarian cancer. A phospho-proteomics screen revealed that PA, which is predominantly produced by diacylglycerol kinase alpha (DGKA) in ovarian cancer, activates the transcription factor c-JUN. DGKA and PA facilitate JNK recruitment to c-JUN and nuclear localization leading to its activation upon cisplatin exposure. Through a genomic screen, we identified that activated c-JUN enhances expression of WEE1 and consequently prevents cells from aberrant cell cycle progression upon cisplatin exposure. DGKA-cJUN-WEE1 signaling was elevated in tumors derived from ovarian cancer patients who did not respond to platinum-based therapy compared to tumors from patients who responded to treatment. Our study demonstrates how lipid metabolism, kinase signaling, and gene regulatory systems are intertwined to direct cisplatin resistance in ovarian cancer. Citation Format: Jie Li, Chaoyun Pan, Austin C. Boese, Anna Umano, Sumin Kang. A DGKA-cJUN-WEE1 signaling axis provides platinum resistance in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4084.

DGKA Provides Platinum Resistance in Ovarian Cancer Through Activation of c-JUN-WEE1 Signaling.

Although platinum compounds are the first-line treatment for ovarian cancer, the majority of patients relapse and develop resistance to treatment. However, the mechanism underlying resistance is unclear. The goal of our study is to decipher the mechanism by which a metabolic kinase, diacylglycerol kinase alpha (DGKA), confers platinum resistance in ovarian cancer. Metabolic kinase RNAi synthetic lethal screening was used to identify a cisplatin resistance driver in ovarian cancer. DGKA variants were used to demonstrate the need for DGKA activity in cisplatin resistance. Phospho-proteomic and genomic screens were performed to identify downstream effectors of DGKA. Therapeutic efficacy of targeting DGKA was confirmed and clinical relevance of DGKA signaling was validated using ovarian cancer patient-derived tumors that had different responses to platinum-based therapy. We found that platinum resistance was mediated by DGKA and its product, phosphatidic acid (PA), in ovarian cancer. Proteomic and genomic screens revealed that DGKA activates the transcription factor c-JUN and consequently enhances expression of a cell-cycle regulator, WEE1. Mechanistically, PA facilitates c-JUN N-terminal kinase recruitment to c-JUN and its nuclear localization, leading to c-JUN activation upon cisplatin exposure. Pharmacologic inhibition of DGKA sensitized ovarian cancer cells to cisplatin treatment and DGKA-c-JUN-WEE1 signaling positively correlated with platinum resistance in tumors derived from patients with ovarian cancer. Our study demonstrates how the DGKA-derived lipid messenger, PA, contributes to cisplatin resistance by intertwining with kinase and transcription networks, and provides preclinical evidence for targeting DGKA as a new strategy in ovarian cancer treatment to battle cisplatin resistance.

Single vector multiplexed shRNA provides a non-gene edited strategy to concurrently knockdown the expression of multiple genes in CAR T cells.

3103 Background: Engineered T cells expressing chimeric antigen receptors (CAR) are now delivering clinically relevant results in patients with advanced hematological malignancies. One critical area for future development is to modulate gene expression thereby endowing the engineered T cell with specific desired features that enhance anti-tumor activity. Methods: Short-hairpin RNA (shRNA) were cloned individually or multiplexed within micro-RNA scaffolds that enabled the co-expression of the individual shRNA with a CAR and a selectable marker all driven by a PolII promoter within a single retroviral vector. Primary human T cells transduced with the CAR-shRNA vectors were selected, expanded in vitro, subjected to negative selection to eliminate any remaining TCR+ cells and examined for target gene expression and functional activity. Results: A 500bp DNA fragment incorporating a shRNA-specific for CD3ζ cloned into a retroviral vectoreffectively knocked down expression of CD3ζ in transduced BCMA-specific CAR T cells. The consequent reduction of cell surface TCR expression resulted in minimal cytokine production upon TCR stimulation in vitro providing a potential allogeneic CAR T approach. These CAR T cells showed no demonstrable evidence of GvHD induction when infused in NSG mice yet maintained BCMA-specific CAR activity in KMS-11 and RPMI-8226 established myeloma models. Initial studies further confirmed that two shRNA could be expressed from a single retroviral vector to modulate the expression of multiple genes. Further engineering of the microRNA framework reduced the size of the transgene load to 394bp while enabling the expression of up to 4 shRNA within a single vector. shRNA specific for CD3ζ, beta-2-microglobulin, CD52 and diacylglycerol kinase alpha were engineered into the framework downstream of a CD19-CAR. Transduced Jurkat cells showed concurrent knockdown of the respective gene products at the mRNA and protein levels. Conclusions: A first-in-human clinical trial evaluating the first-generation single shRNA-vector in the context of a BCMA-targeting CAR as a non-gene edited approach to allogeneic CAR T cell therapy will be initiated in 2020. The proof of principle study here shows that multiple shRNAs are active within a single viral vector thereby avoiding the need for bespoke individual clinical reagents to target multiple genes. The multiplexed shRNA vector system is now in further development to explore whether this strategy can enhance the therapeutic potential of CAR T cells.

Diacylglycerol Kinase Alpha in Radiation-Induced Fibrosis: Potential as a Predictive Marker or Therapeutic Target

Radiotherapy is an efficient tool in cancer treatment, but it brings along the risk of side effects such as fibrosis in the irradiated healthy tissue thus limiting tumor control and impairing quality of life of cancer survivors. Knowledge on radiation-related fibrosis risk and therapeutic options is still limited and requires further research. Recent studies demonstrated that epigenetic regulation of diacylglycerol kinase alpha (DGKA) is associated with radiation-induced fibrosis. However, the specific mechanisms are still unknown. In this review, we scrutinized the role of DGKA in the radiation response and in further cellular functions to show the potential of DGKA as a predictive marker or a novel target in fibrosis treatment. DGKA was reported to participate in immune response, lipid signaling, exosome production, and migration as well as cell proliferation, all processes which are suggested to be critical steps in fibrogenesis. Most of these functions are based on the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) at plasma membranes, but DGKA might have also other, yet not well-known functions in the nucleus. Current evidence summarized here underlines that DGKA activation may play a central role in fibrosis formation post-irradiation and shows a potential of direct DGKA inhibitors or epigenetic modulators to attenuate pro-fibrotic reactions, thus providing novel therapeutic choices.

Diacylglycerol kinase alpha (DGKalpha) contribution to CD4+ T cell plasticity

Diacylglycerol kinase alpha (DGKalpha) contribution to CD4+ T cell plasticity

Inhibition of diacylglycerol kinase alpha to augment antitumor effector T cells in tumor-bearing host.

293 Background: Diacylglycerol kinases (DGKs), lipid kinases transforming diacylglycerol to phosphatidic acid, play important roles in intracellular signal transduction. Diacylglycerol kinase alpha (DGKa), an isozyme of DGKs, is well-known to promote proliferation of cancer cells by suppression of the apoptosis. Additionally, a previous report demonstrated that activation of DGKa induced anergy state of T lymphocytes in vivo. In this study, we investigated whether inhibition of DGKa not only suppress the tumorigenesis of cancer cells but also activate anti-tumor immunity. Methods: We first investigated the effect of DGKa inhibitor on in vitro proliferation of murine hepatoma cell lines (Hepa1-6) by cell proliferation assay. Cytokine and Granzyme B productions by CD8+ T cells from OT-1 mice after the OVA antigen stimulation were evaluated by ELISA and flowcytometry, respectively. Next, we established a tumor-bearing mice model by injection of mCherry-transfected Hepa1-6 cells into spleen. Tumorigenesis and tumor-infiltrating T cells in the liver were evaluated by in vivo imaging system, HE staining, and immunohistochemistry. CD8+ T cells were collected from the liver and stimulated with PMA and Ca2+ ionophore and the IFN-g production levels were evaluated by flowcytometry. Results: Proliferation of Hepa1-6 cells were suppressed in the presence of DGKa inhibitor in vitro. IL-2 production levels of OT-1 CD8 T cells in control group was augmented by the addition of DGKa inhibitor (246 vs 579 pg/ml, p &lt; 0.05). Granzyme B-positive cells in OT-1 CD8+ T cells were increased by the treatment with DGKa inhibitor compared to the control group (4.4 vs 8.9 %, P &lt; 0.05) after the antigen stimulation. In vivo administration of DGKa inhibitor significantly suppressed the tumor size (fluorescence (AU) 2.0x1010 vs 6.3x109, area (μm2) 1.5x107 vs 0.9x107, p &lt; 0.05) in the liver of tumor bearing mice. Then, the number of tumor-infiltrating T cells (582 vs 1506, 5 HPF, p &lt; 0.05) and the IFN-g-producing cells (9.2 vs 16.0 %) in CD8+ T cells were elevated by the DGKa treatment. Conclusions: Inhibition of DGKa not only suppressed the proliferation of hepatoma but also activated anti-tumor effector T cells in vivo.

Identification of a novel DGKα inhibitor for XLP-1 therapy by virtual screening

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an in-silico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.

ILDR2 has a negligible role in hepatic steatosis.

We have previously reported that Ildr2 knockdown via adenovirally-delivered shRNA causes hepatic steatosis in mice. In the present study we investigated hepatic biochemical and anatomic phenotypes of Cre-mediated Ildr2 knock-out mice. Liver-specific Ildr2 knock-out mice were generated in C57BL/6J mice segregating for a floxed (exon 1) allele of Ildr2, using congenital and acute (10-13-week-old male mice) Cre expression. In addition, Ildr2 shRNA was administered to Ildr2 knock-out mice to test the effects of Ildr2 shRNA, per se, in the absence of Ildr2 expression. RNA sequencing was performed on livers of these knockdown and knockout mice. Congenital and acute liver-specific and hepatocyte-specific knockout mice did not develop hepatic steatosis. However, administration of Ildr2 shRNA to Ildr2 knock-out mice did cause hepatic steatosis, indicating that the Ildr2 shRNA had apparent “off-target” effects on gene(s) other than Ildr2. RNA sequencing and BLAST sequence alignment revealed Dgka as a candidate gene mediating these “off-target” effects. Ildr2 shRNA is 63% homologous to the Dgka gene, and Dgka expression decreased only in mice displaying hepatic steatosis. Dgka encodes diacylglycerol kinase (DGK) alpha, one of a family of DGKs which convert diacylglycerides to phosphatidic acid for second messenger signaling. Dgka knockdown mice would be expected to accumulate diacylglyceride, contributing to the observed hepatic steatosis. We conclude that ILDR2 plays a negligible role in hepatic steatosis. Rather, hepatic steatosis observed previously in Ildr2 knockdown mice was likely due to shRNA targeting of Dgka and/or other “off-target” genes. We propose that the gene candidates identified in this follow-up study may lead to identification of novel regulators of hepatic lipid metabolism.