Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: BMS-986408 is a potent DGKα and DGKζ lipid kinase inhibitor and degrader. A, Chemical structure of BMS-986408. B, Plots showing the inhibitory dose–response curves for BMS-986408 in recombinant DGKα and DGKζ biochemical lipid kinase assays and corresponding IC 50 values. C, Conversion of D4-Oloeyl-DAG to D4-Oleoyl PA in Jurkat cells treated with 0.25 μmol/L of BMS-986408. Data are represented as the mean ± SD; n = 3 per group. D, Schematic of the BMS-986408 NanoBRET target engagement assay in live cells. E, Time lapse of milliBRET (mBRET) ratio with the BMS-986408-NB590 tracer in DGKα-NanoLuc–overexpressing and NanoLuc-DGKζ–overexpressing cells with (■) or without ( ) saturating unlabeled BMS-986408 (20 μmol/L) to normalize for specificity (top) and DGKi-NB590 binding kinetics to DGKα-NanoLuc and NanoLuc-DGKζ (bottom). Binding affinity is presented in K d ; Data are represented as the mean ± SD; n = 2 per group. F, CETSA melting curves of DGKα (top) and DGKζ (bottom) from Jurkat cells treated with ( ) or without (●) 0.5 μmol/L BMS-986408. Data show the percent change from the 37°C baseline. G, Representative images showing the subcellular localization of YFP-tagged DGKα or DGKζ with or without BMS-986408 (0.25 μmol/L). YFP is colored in green, and nuclear staining is colored in blue. H, Quantification of BMS-986408–induced DGKα ( ) and DGKζ (◆) plasma membrane translocation with half-maximal efficacious concentrations (EC 50 ). I, Degradation dose–response for DGKα and DGKζ in human PBMCs treated with BMS-986408 for 24 hours. β-actin is presented as a loading control. J, Rescue of BMS-986408-mediated degradation with proteosome (bortezomib, BZ) and ubiquitination (TAK-243, E1i) inhibitors. K, Schematic of the whole blood DGKi potency assay, highlighting phospho-ERK and IL2 pharmacodynamic biomarkers. L, Flow cytometry quantification of BMS-986408 phospho-ERK induction potency in whole blood T cells. The EC 50 value is shown for CD4 + (●) and CD8 + ( ) T cells. Data are represented as the mean ± SD.; n = 11 per group. M, AlphaLISA quantification of BMS-986408 IL2 production from human whole blood from two donors. The EC 50 value is shown for each donor. ( D and K, Created with BioRender.com .)

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: Recombinant, Drug discovery, Binding Assay, Staining, Clinical Proteomics, Membrane, Translocation Assay, Control, Ubiquitin Proteomics, Potency Assay, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: BMS-986408 binds with the accessory region of DGKα and DGKζ lipid kinase domain. A, Schematic of the CRISPR base editing screen to select for DGKA or DGKZ mutations that conferred resistance to BMS-986408–mediated degradation of eGFP-DGKα or mNeonGreen-DGKζ expressed in Jurkat cells. B and C, Scatterplot of Log 2 fold change (LFC) sgRNA enrichment in DGKA and DGKZ adenine base editor scanning screens. The dotted line indicates LFC = 1.5, and validated hits are highlighted in green. AlphaFold models of DGKα and DGKζ are shown as ribbons, with C-alpha atoms of enriched residues shown as spheres. The surfaces of the docked ligands (see “Materials and Methods”) are shown to highlight the proposed binding sites. D, Validation of the base editing CRISPR screen using KI cell clones: Jurkat eGFP-DGKα cells harboring the S532P, L556P, or H606R mutations and mNeonGreen-DGKζ cells harboring the F463S, S490P, or C534R mutations were treated with BMS-986408 (0.75 μmol/L), and fluorescence signal was quantified as the mean fluorescence intensity (MFI). Each point represents a cell clone. E, BMS-986408 CETSA dose–response at 41.5°C showing that HiBit-tagged DGKα harboring the S532P, L556P, or H606R mutations was resistant to BMS-986408–mediated thermal destabilization. F, BMS-986408 CETSA dose–response at 43.1°C showing that HiBit-tagged DGKζ harboring the F463S, S490P, or C534R mutations was resistant to BMS-986408–mediated thermal destabilization. G, Closer view of docked poses with enriched residues’ side chains shown as orange sticks and validated residues’ side chains shown as green sticks. H, Electrostatic surface representation of the proposed binding sites, with the surfaces of validated residues shown in green. WT, wild-type. ( A, Created with BioRender.com .)

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: CRISPR, Binding Assay, Biomarker Discovery, Clone Assay, Fluorescence

Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: Dual DGKα/ζ inhibitor BMS-986408 unleashes PD-1 T-cell checkpoint therapy. A, Schematic of TCR signaling cascade, with TCR and CD28 providing positive signals and PD-1 and DGKα/ζ providing negative signals. B, Therapeutic efficacy of anti–PD-1, BMS-986408, or the combination therapy in SA1N, MC38, and CT26 tumor models. Each line represents tumor volume of one individual animal. n = 10 per group. The percentage of animals achieving CR is noted on each plot. C, Heatmap of differentially expressed genes from RNA-seq data from MC38 tumors at day 7 after treatment. The black and grey barcodes indicates whether the expression change is statistically different between the vehicle and combination treatment group. D, Volcano plots of RNA-seq data from the same analysis. Upregulated genes are highlighted in purple, and downregulated genes in blue; a subset of upregulated T-cell effector genes are labeled in each plot. E, Flow cytometry quantification of granzyme B+ and Ki67+ effector CD8 + populations in the MC38 tumors. Data were collected at day 7 after treatment initiation. F, Flow cytometry quantification of naïve (CD44 − CD62L + ), effector/effector memory (E/EM; CD44 + CD62L − ), central memory (CM; CD44 + CD62L + ), and activated (CD69 + ; PD-1+ or Ki67+) CD8 + T-cell subsets in MC38 TdLNs. Data were collected at day 7 after treatment initiation. G, Flow cytometric quantification of GFP+ CD8 + T cells in the TdLN of MC38 tumors implanted into Nur77-GFP transgenic mice. Data were collected 24 hours after treatment with anti–PD-1, BMS-986408, or the combination. H, In vivo priming of tumor antigen–specific T cells. TRP1 high or TRP1 low transgenic CD8 + T cells were labeled with CTV and adoptively transferred into mice implanted with C2VTrp1 tumors. Mice were dosed with anti–PD-1, BMS-986408, or the combination treatment. Representative flow cytometry analysis of CTV dilution in adoptively transferred cells is shown. Gates delineate different generations of proliferated cells. I, Calculated proliferation index (see “Materials and Methods”) of adoptively transferred TRP1 High and TRP1 Low CD8 + T cells in the TdLN 5 days after treatment with either anti–PD-1, BMS-986408, or the combination therapy; n = 5 per group. Error bars represent the SD. Statistical analysis was performed using an ordinary one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Combo, combination; Ctrls, controls; SSC, side scatter.

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: Drug discovery, RNA Sequencing, Expressing, Labeling, Flow Cytometry, Transgenic Assay, In Vivo

Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: Inhibiting both DGKα and DGKζ maximizes anti–PD-1 combination benefit. A, Therapeutic efficacy of anti–PD-1 vs. the combination of anti–PD-1 with either BMS-986408, DGKα-i, DGKζ-i, or DGKα-i + DGKζ-i in the MC38 tumor model. Each line represents the tumor volume curve from one individual animal. The percentage of animals achieving CR is noted on each plot. B, Cytotoxicity evaluation of NY-ESO-1–specific effector T cells in the presence of DGKα-i, DGKζ-i, or BMS-408. All compounds were dosed at 0.1 μmol/L; n = 6 per group. C, Proliferation of human PBMCs (left) and mouse TRP1 high T cells (right) in the presence of DGKα-i, DGKζ-i, or BMS-408. All compounds were dosed at 0.1 μmol/L; n = 5 per group. D, In vivo proliferation indices of adoptively transferred TRP1 high T cells in recipient mice dosed with DGKα-i, DGKζ-i, or BMS-408; n = 5 per group. E and F, Human PBMC proliferation and IFNγ production in a matrixed combination dose–response of DGKα-i or DGKζ-i (left) with corresponding highest single agent (HSA) synergy analysis (right); n = 6 per group. G, Heatmap of phosphopeptides significantly changed in human T cells treated with dose titrations of DGKα-i, DGKζ-i, or BMS-986408 from 0.001 to 1 μmol/L. Values represent the signed effect size of the dose–response curves (see “Materials and Methods”), with purple showing increased phosphorylation and blue showing decreased phosphorylation. H, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of significantly increased phosphoproteins from ( G ). Top 10 pathways with −log 10 (FDR) is presented. I, Dose effect sizes of selected phosphopeptides from the NF-κB pathway and MAPK pathway as in ( G ). Error bars represent the SD. Statistical analysis was performed using an ordinary one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Ctrls, controls.

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: Drug discovery, In Vivo, Phospho-proteomics

Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: Translational data supporting the combination of DGKα/ζ and PD-1 inhibitors in NSCLC. A, Schematic of the translational research strategy to evaluate DGKα/ζ expression and inhibition in NSCLC patient tumor biopsies. B, Uniform Manifold Approximation and Projection (UMAP) plot of scRNA-seq data from patients with NSCLC (see “Materials and Methods”). Immune cell populations were plotted and color-coded by their corresponding signature gene expression. Right shows the expression overlay of genes of interest. C, Representative multiplexed immunofluorescence images showing the expression of CD3, CD4, CD8, PD-1, DGKα, and DGKζ in a NSCLC patient tumor biopsy. D, Dot plot summary of DGKα, DGKζ, and several additional immune checkpoint expression in NSCLC TIL subsets from 78 patients with NSCLC. Dot sizes represent log 2 cell count, and dot colors represent log 2 mIF intensity. E, Cytokine quantification in the PDOTS cultures with anti–PD-1, BMS-408, or combination treatment. F, Absolute quantification of IFNγ release in PDOTS cultures, grouped by each individual patients and further divided into responders/nonresponders based on whether anti–PD-1 and BMS-408 combination induced significant increase of IFNγ release. Statistical analysis was performed using an ordinary one-way ANOVA. Error bars represent the SD. G, Tumor mutation burden of PDOTS tumors grouped by IFNγ responder status. Student t test was performed between the two groups. In all studies, data were collected 3 days after treatment, and BMS-986408 was dosed at 0.3 μmol/L. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001. Ctrls, controls; mIF, multiplexed immunofluorescence; Pt, patient. ( A, Created with BioRender.com .)

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: Clinical Proteomics, Expressing, Inhibition, Gene Expression, Immunofluorescence, Cell Counting, Quantitative Proteomics, Mutagenesis

Journal: Cancer Immunology Research

Article Title: Discovery of BMS-986408, a First-In-Class Dual DGKα and DGKζ Inhibitor that Unleashes PD-1 Checkpoint and CAR T-cell Immunotherapies

doi: 10.1158/2326-6066.CIR-25-0156

Figure Lengend Snippet: Dual DGKα/ζ inhibitor BMS-986408 unleashes CAR T-cell therapy. A, Growth curves of Raji transduced with red-shifted firefly luciferase (Raji-rFluc) tumor in NOD/SCID gamma mice over time. Mice were given a suboptimal dose of 1 × 10 6 CAR-T cells of different genotypes and dosed with or without 0.3 mpk BMS-986408. B and C, Modified tumor control index (see “Materials and Methods”) and CAR-T cells per μL blood from each group. Nonparametric Kruskal–Wallis test was performed followed by the Benjamini, Krieger, and Yekutieli FDR correction for multiple comparisons. D and E, Chronically stimulated CAR T cells (CAR-T) were removed from plate-bound stimulus and plated with A549.CD19 or Granta-519 3D spheroids with varying treatment levels of BMS-986408. Normalized tumor area (RCU μm 2 ) was assessed on day 9. Friedman test was performed with Dunn post hoc test for multiple comparisons. *, P < 0.05; **, P < 0.01. F, Growth curves of Nalm6 transduced with red-shifted firefly luciferase (Nalm6-rFluc) tumor in NOD/SCID gamma mice over time. Mice were dosed with BMS-986408 (0.3 mpk), 1 × 10 6 CAR-T, or the combination of both. G, Nalm6-rFluc tumor growth curves were analyzed calculated as modified tumor control index. Student t test was performed between the two groups. H, Blood circulating CAR-T were quantified by flow cytometry on days 8, 16, 23, and 30. For all plots, error bars represent the SD. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: A recombinant DGKα (SignalChem, D21-10BH) DAG phosphorylation assay was established using the PhosphoSens kinase assay platform (AssayQuant).

Techniques: Transduction, Luciferase, Modification, Control, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: CAM12203 directly binds to DGK ζ . (A) Structure of the biotinylated probe. (B) RAW264.7 cells (pretreated with or without the probe for 2 h) were stimulated with LPS for 4 h. The mRNA level of Il1b was detected by Q-PCR. (C) BMDMs (pretreated with or without the probe for 2 h) were stimulated with LPS (4 h) + ATP (another 45 min). The IL-1 β concentration in culture medium (CM) was examined by HTRF. (D) Overall scheme of the experiments to identify the targets of CAM12203 (created with Biorender.com ). (E) The list of gene names of CAM12203 -binding proteins. (F) RAW264.7 lysate was pretreated with or without CAM12203 for 2 h (4 °C), followed by incubation with the probe for another 2 h (4 °C). The level of DGK ζ pulled down was determined by Western blotting. (G) RAW264.7 lysate was treated with or without CAM12203 , and the protein stability of DGK ζ resistant to pronase E was determined by DARTS. (H) RAW264.7 lysate was treated with or without CAM12203 , and the thermostability of DGK ζ was determined by CETSA. (I) DGK ζ protein was treated with CAM12203 , and the binding affinity was determined by SPR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Concentration Assay, Binding Assay, Incubation, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ plays a critical role in macrophage-mediated inflammation and liver failure. (A) The protein level of DGK ζ in LPS-stimulated RAW264.7 cells was determined by Western blotting. (B–D) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS. The level of PA (B) and IL-1 β expression (C, D) were measured by a commercial kit, Q-PCR, and Western blotting, respectively. (E) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The IL-1 β expression was detected by Q-PCR and Western blotting. (F–H) BMDMs from wild-type (WT) and Dgkz −/− mice were stimulated with LPS + ATP. The culture medium (CM) was collected for examination of IL-1 β concentration (F) by HTRF, and added to the primary hepatocytes along with D-GalN. The levels of cleaved-PARP (c-PARP) and cleaved-Caspase 3 (c-Caspase 3) (G), and the activity of Caspase 3 (scar bar: 100 μm) (H) were determined. (I, J) ALF was induced in mice by LPS + D-GalN. After 1.5, 3 and 4 h, the protein level of DGK ζ in the liver was determined by Western blotting (I). After 6 h, the protein levels of DGK ζ in F4/80 + and Ly6G + cells sorted from the liver were determined by Western blotting (J). (K) Hepatic DGK ζ + CD68 + cells in Healthy Controls ( n = 6) and ACLF patients ( n = 5) were marked (scar bar: 50 μm) and counted. (L–N) WT and Dgkz −/− mice were administered with LPS + D-GalN for 6 h. The liver index (L), the levels of ALT, AST and LDH in plasma (M), the histological alterations of the liver (scar bar: 50 μm) (N), the frequency of MoMFs in liver (O), the mRNA level of Il1b in liver (P), and the IL-1 β concentration in plasma (Q) were evaluated. Data are displayed as mean ± SEM (For A–H, n = 3; For I, J, I–N, n = 6). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Western Blot, Expressing, Transfection, Concentration Assay, Activity Assay, Clinical Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ –STAT3 axis drives IL-1 β production in macrophages. (A–C) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for the indicated time. The levels of pp65, pSTAT3 Ser727 , and pSTAT3 Tyr705 , as well as the nuclear translocation of STAT3 were determined by Western blotting. (D) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (E) Regulators of the JAK2–STAT3 pathway (created with Biorender.com ). (F, G) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for 4 h. The levels of pJAK2 (F), SOCS1, and SOCS3 (G) were determined by Western blotting. (H) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without TPI-1 (3 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 were determined by Western blotting. (I) RAW264.7 cells (with or without U73122 (10 μmol/L), 2-APB (50 μmol/L), and BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 as well as IL-1 β expression were determined by Western blotting and Q-PCR. (J, K) RAW264.7 cells (with or without BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The binding between Pyk2 and JAK2 (J) was examined by immunoprecipitation, and the levels of pPyk2 and pJAK2 (K) were determined by Western blotting. (L, M) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS as well as PA for 4 h. The concentration of Ca 2+ (L) was measured by Fluo-4 AM (scar bar: 50 μm). The levels of pPyk2, pJAK2, and pSTAT3 Tyr705 as well as IL-1 β expression (M) were determined by Western blotting and Q-PCR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Translocation Assay, Western Blot, Transfection, Expressing, Binding Assay, Immunoprecipitation, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: The anti-inflammatory effect of CAM12203 relies on DGK ζ . (A) Overall scheme of the examination of DGK ζ activity (created with Biorender.com ). (B) The effects of BAY 2965501 (BAY) and CAM12203 on DGK ζ activity were measured. (C) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 0.25 h. The level of PA was measured by a commercial kit. (D–F) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The levels of pPyk2, pJAK2 (D) and pSTAT3 Tyr705 (E), and the cytosol as well as nuclear levels of STAT3 (F) were determined by Western blotting. (G) PMA-differentiated THP-1 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (H, I) Mice were administered by CAM12203 (30 mg/kg), and ALF was induced by LPS + D-GalN. After 6 h, the levels of pPyk2, pJAK2 (H), and pSTAT3 Tyr705 (I) in F4/80 + cells sorted from the liver were determined by Western blotting. (J–L) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without CAM12203 /BAY pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 and IL-1 β expression were determined by Western blotting and Q-PCR. (M) RAW264.7 cells were transfected with Control or DGK ζ -OE plasmid, and treated with or without CAM12203 /BAY for 2 h, followed by LPS simulation for 4 h. The levels of pSTAT3 Tyr705 and pro-IL-1 β were determined by Western blotting. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group; ns, not significant.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Control, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: CAM12203 directly binds to DGK ζ . (A) Structure of the biotinylated probe. (B) RAW264.7 cells (pretreated with or without the probe for 2 h) were stimulated with LPS for 4 h. The mRNA level of Il1b was detected by Q-PCR. (C) BMDMs (pretreated with or without the probe for 2 h) were stimulated with LPS (4 h) + ATP (another 45 min). The IL-1 β concentration in culture medium (CM) was examined by HTRF. (D) Overall scheme of the experiments to identify the targets of CAM12203 (created with Biorender.com ). (E) The list of gene names of CAM12203 -binding proteins. (F) RAW264.7 lysate was pretreated with or without CAM12203 for 2 h (4 °C), followed by incubation with the probe for another 2 h (4 °C). The level of DGK ζ pulled down was determined by Western blotting. (G) RAW264.7 lysate was treated with or without CAM12203 , and the protein stability of DGK ζ resistant to pronase E was determined by DARTS. (H) RAW264.7 lysate was treated with or without CAM12203 , and the thermostability of DGK ζ was determined by CETSA. (I) DGK ζ protein was treated with CAM12203 , and the binding affinity was determined by SPR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Concentration Assay, Binding Assay, Incubation, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ plays a critical role in macrophage-mediated inflammation and liver failure. (A) The protein level of DGK ζ in LPS-stimulated RAW264.7 cells was determined by Western blotting. (B–D) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS. The level of PA (B) and IL-1 β expression (C, D) were measured by a commercial kit, Q-PCR, and Western blotting, respectively. (E) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The IL-1 β expression was detected by Q-PCR and Western blotting. (F–H) BMDMs from wild-type (WT) and Dgkz −/− mice were stimulated with LPS + ATP. The culture medium (CM) was collected for examination of IL-1 β concentration (F) by HTRF, and added to the primary hepatocytes along with D-GalN. The levels of cleaved-PARP (c-PARP) and cleaved-Caspase 3 (c-Caspase 3) (G), and the activity of Caspase 3 (scar bar: 100 μm) (H) were determined. (I, J) ALF was induced in mice by LPS + D-GalN. After 1.5, 3 and 4 h, the protein level of DGK ζ in the liver was determined by Western blotting (I). After 6 h, the protein levels of DGK ζ in F4/80 + and Ly6G + cells sorted from the liver were determined by Western blotting (J). (K) Hepatic DGK ζ + CD68 + cells in Healthy Controls ( n = 6) and ACLF patients ( n = 5) were marked (scar bar: 50 μm) and counted. (L–N) WT and Dgkz −/− mice were administered with LPS + D-GalN for 6 h. The liver index (L), the levels of ALT, AST and LDH in plasma (M), the histological alterations of the liver (scar bar: 50 μm) (N), the frequency of MoMFs in liver (O), the mRNA level of Il1b in liver (P), and the IL-1 β concentration in plasma (Q) were evaluated. Data are displayed as mean ± SEM (For A–H, n = 3; For I, J, I–N, n = 6). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Western Blot, Expressing, Transfection, Concentration Assay, Activity Assay, Clinical Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: DGK ζ –STAT3 axis drives IL-1 β production in macrophages. (A–C) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for the indicated time. The levels of pp65, pSTAT3 Ser727 , and pSTAT3 Tyr705 , as well as the nuclear translocation of STAT3 were determined by Western blotting. (D) PMA-differentiated THP-1 cells were transfected with siNC or si DGKZ , followed by LPS stimulation for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (E) Regulators of the JAK2–STAT3 pathway (created with Biorender.com ). (F, G) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS for 4 h. The levels of pJAK2 (F), SOCS1, and SOCS3 (G) were determined by Western blotting. (H) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without TPI-1 (3 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 were determined by Western blotting. (I) RAW264.7 cells (with or without U73122 (10 μmol/L), 2-APB (50 μmol/L), and BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The levels of pJAK2 and pSTAT3 Tyr705 as well as IL-1 β expression were determined by Western blotting and Q-PCR. (J, K) RAW264.7 cells (with or without BAPTM-AM (10 μmol/L) pretreatment for 30 min) were stimulated with LPS for 4 h. The binding between Pyk2 and JAK2 (J) was examined by immunoprecipitation, and the levels of pPyk2 and pJAK2 (K) were determined by Western blotting. (L, M) Stable shNC- and sh Dgkz -RAW264.7 cells were stimulated with LPS as well as PA for 4 h. The concentration of Ca 2+ (L) was measured by Fluo-4 AM (scar bar: 50 μm). The levels of pPyk2, pJAK2, and pSTAT3 Tyr705 as well as IL-1 β expression (M) were determined by Western blotting and Q-PCR. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Translocation Assay, Western Blot, Transfection, Expressing, Binding Assay, Immunoprecipitation, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage DGK ζ -mediated phosphatidic acid remodeling aggravates acute liver failure

doi: 10.1016/j.apsb.2025.06.019

Figure Lengend Snippet: The anti-inflammatory effect of CAM12203 relies on DGK ζ . (A) Overall scheme of the examination of DGK ζ activity (created with Biorender.com ). (B) The effects of BAY 2965501 (BAY) and CAM12203 on DGK ζ activity were measured. (C) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 0.25 h. The level of PA was measured by a commercial kit. (D–F) RAW264.7 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The levels of pPyk2, pJAK2 (D) and pSTAT3 Tyr705 (E), and the cytosol as well as nuclear levels of STAT3 (F) were determined by Western blotting. (G) PMA-differentiated THP-1 cells (with or without CAM12203 pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 was determined by Western blotting. (H, I) Mice were administered by CAM12203 (30 mg/kg), and ALF was induced by LPS + D-GalN. After 6 h, the levels of pPyk2, pJAK2 (H), and pSTAT3 Tyr705 (I) in F4/80 + cells sorted from the liver were determined by Western blotting. (J–L) Stable shNC- and sh Dgkz -RAW264.7 cells (with or without CAM12203 /BAY pretreatment for 2 h) were stimulated with LPS for 4 h. The level of pSTAT3 Tyr705 and IL-1 β expression were determined by Western blotting and Q-PCR. (M) RAW264.7 cells were transfected with Control or DGK ζ -OE plasmid, and treated with or without CAM12203 /BAY for 2 h, followed by LPS simulation for 4 h. The levels of pSTAT3 Tyr705 and pro-IL-1 β were determined by Western blotting. Data are displayed as mean ± SEM ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01 vs . the indicated group; ns, not significant.

Article Snippet: Primary antibodies applied included DGK ζ (1:1000; ab239080) which was obtained from Abcam (Cambridge, UK), proline rich tyrosine kinase 2 (Pyk2) (1:3000; 17592-1-AP), DGK α (1:2000; 11547-1-AP), DGK ε (1:1000; 11900-1-AP), DGK θ (1:500; 17885-1-AP) and β -Actin (1:8000; 66009-1-Ig) which were obtained from Proteintech (Wuhan, China), DGK γ (1:500; DF13919) which was obtained from Affinity (Cincinnati, USA), DGK δ (1:500; A15115) and α -Tubulin (1:1000; A6830) which were obtained from Ablconal, IL-1 β (1:1000; 12242), poly-ADP-ribose-polymerase (PARP) (1:1000; 9542), Caspase 3 (1:1000; 9662), p65 (1:1000; 8242), pp65 (1:1000; 3033), STAT3 (1:1000; 9139), pSTAT3 Ser727 (1:1000; 9134), pSTAT3 Tyr705 (1:2000; 9145), and pPyk2 (1:1000; 3291) which were obtained from Cell Signaling Technology (Danvers, USA), janus kinase 2 (JAK2) (1:1000; AF1489) and pJAK2 (1:500; AF5854) which were obtained from Beyotime, suppressor of cytokine signaling-1 (SOCS1) (1:500; WL05128) and SOCS3 (1:1000; WL01364) which were obtained from Wanleibio (Shenyang, China).

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Control, Plasmid Preparation

Journal: Molecular Therapy Oncology

Article Title: CRISPR-Cas9 knockout of DGKα/ζ improves the anti-tumor activities of TAG-72 CAR-T cells in ovarian cancer

doi: 10.1016/j.omton.2025.200962

Figure Lengend Snippet: Characterization of TAG-72 CAR/DGKα/ζ KO T cells (A) Overview of the process used to produce CAR-T cells. (B) Schematic of the lentiviral expression cassette containing an anti-TAG-72-specific scFv, CD8 hinge domain, and 4-1BB intracellular co-stimulatory domain linked to a CD3ζ signaling domain. (C) Transduction efficiency of the TAG-72 CAR was assessed using flow cytometry at least 11 days after transduction. Unedited T cells were included as a control. Representative of 3 independent experiments. (D) Frequency of total indels and KO (proportion of indels that indicate frameshift mutations) of DGKα and DGKζ genes in the pooled gene-edited CAR-T cells as assessed by ICE analysis ( n = 3 independent donors). (E) Western blots of DGKα and DGKζ protein in TAG-72 CAR/DGKα/ζ KO CAR-T cell lysates and unedited control T cells (NT). β-actin was used as a loading control. (F) Flow cytometry analysis was performed to examine the common T cell-associated surface markers CD4 and CD8 and effector memory (CCR7 − /CD45RO + ), central memory (CCR7 + /CD45RO + ), naive (CCR7 + /CD45RO − ), and effector (CCR7 − /CD45RO − ) cell subsets and exhaustion markers (as indicated). Frequencies are presented as a proportion of viable cells ( n = 3–4 independent donors). A Kruskal-Wallis non-parametric test confirmed no significant differences between groups.

Article Snippet: Antibodies against DGKα (Santa Cruz Biotechnology, Dallas, TX, USA) and DGKζ (Sigma-Aldrich) were used as probes.

Techniques: Expressing, Transduction, Flow Cytometry, Control, Western Blot

Journal: Molecular Therapy Oncology

Article Title: CRISPR-Cas9 knockout of DGKα/ζ improves the anti-tumor activities of TAG-72 CAR-T cells in ovarian cancer

doi: 10.1016/j.omton.2025.200962

Figure Lengend Snippet: Levels of secreted cytokines following co-culture Shown are (A) pro-inflammatory/regulatory cytokines, (B) anti-inflammatory cytokines, (C) chemotactic factors, and (D) growth factors in supernatants harvested from unedited T cells (blue) and TAG-72 CAR-T (red) and TAG-72 CAR/DGKα/ζ KO T cells (purple) co-cultured with the TAG-72 m i d cancer cell line OVCAR-3 for 20 h. Biological and intra-assay triplicates were used and are denoted as mean ± SD ( n = 3). A one-way ANOVA with Tukey’s multiple comparison test was used to determine statistical significance. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Antibodies against DGKα (Santa Cruz Biotechnology, Dallas, TX, USA) and DGKζ (Sigma-Aldrich) were used as probes.

Techniques: Co-Culture Assay, Cell Culture, Intra Assay, Comparison

Journal: Molecular Therapy Oncology

Article Title: CRISPR-Cas9 knockout of DGKα/ζ improves the anti-tumor activities of TAG-72 CAR-T cells in ovarian cancer

doi: 10.1016/j.omton.2025.200962

Figure Lengend Snippet: Impact of CAR-T cells on the in vivo growth of established OVCAR-3 tumors (A and B) NSG mice bearing OVCAR-3 tumors were treated at 5-day intervals (indicated by dashed lines) with either 5 × 10 6 unedited T cells (red), TAG-72 CAR-T cells (orange), TAG-72 CAR/DGKα single KO T cells (black), TAG-72 CAR/DGKζ single KO T cells (green), or TAG-72 CAR/DGKα/ζ KO T cells (blue) by intravenous (i.v.) injection when the starting tumor volume (STV) was approximately 150 mm 3 in size ( n = 8–23 mice per group, results pooled from 5 independent experimental cohorts where animals reached STV size, on average, in 8 weeks). (C) Kaplan-Meier survival curves of all treatment groups. Paired log rank (Mantel-Cox) tests were used to determine statistical significance with probabilities as shown. (D) Representative IHC of OVCAR-3 tumors in NSG xenograft mice. Blue scale bar, 2 mm; black scale bar, 200 μm.

Article Snippet: Antibodies against DGKα (Santa Cruz Biotechnology, Dallas, TX, USA) and DGKζ (Sigma-Aldrich) were used as probes.

Techniques: In Vivo, Injection