compound 78c  (TargetMol)

Journal: Translational Oncology

Article Title: CD38 contributes to tumor progression and tumor microenvironment reshaping in epithelial ovarian cancer

doi: 10.1016/j.tranon.2025.102414

Figure Lengend Snippet: Evaluating CD38’s impact on cell infiltration and communication in ovarian cancer by single-cell resolution (A) Cell type annotation for six datasets categorized by major lineage. The heatmap showed the average gene expression of CD38 in different populations of malignant, immune and stromal cells. (B) UMAP dimensionality reduction of cellular landscape of ovarian cancer and CD38 expression across different cell populations. (C) Violin plots illustrated the distribution of CD38 expression levels among various cell populations in normal and ovarian cancer tissues. (D) Violin plots illustrated CD38 expression patterns among cell populations in primary, metastatic, and relapsed ovarian cancer tissues. (E) Heatmap showed the intensity of intercellular communication in the GSE118828 dataset. (F) Comparison of gene expression levels involved in cell-cell interactions between high and low CD38-expressing groups in the GSE9891 dataset (* p < 0.05, ** p < 0.01, **** p < 0.0001). (G) qPCR analysis of relative mRNA expression of cell-cell interaction genes in subcutaneous tumors from C57BL/6 mice treated with Compound 78c, normalized to β-actin ( n = 3 per group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Four weeks post-inoculation, the CD38-specific inhibitor Compound 78c (S8906, Selleck Chemicals, TX, USA) was administered to C57BL/6 by intraperitoneal injection (10 mg/kg/dose [ ]) every two days over a period of 3 weeks.

Techniques: Gene Expression, Expressing, Comparison

Journal: Translational Oncology

Article Title: CD38 contributes to tumor progression and tumor microenvironment reshaping in epithelial ovarian cancer

doi: 10.1016/j.tranon.2025.102414

Figure Lengend Snippet: CD38 promotes ovarian cancer proliferation, metastasis and immune cell infiltration in vivo (A) Tumor formation in C57BL/6 mice ubcutaneously injected with ID8 cells stably expressing vector or CD38 (ID8-Vector or ID8-OVE-CD38). After 6 weeks, tumors were excised and photographed. (B) Quantification of tumor volume and total tumor weight in the transplanted tumors ( n = 7 for ID8-Vector, n = 5 for ID8-OVE-CD38, *p<0.05, *** p < 0.001). (C) Flow cytometry detected T cell proportion in the transplanted tumors ( n = 3 per group, * p < 0.05). (D) Abdominal metastases in BALB/c nude mice intraperitoneally injected with SKOV3 cells stably expressing vector or CD38 (SKOV3-Vector or SKOV3-OVE-CD38). After 4 weeks, tumors were excised and photographed. Upper panels: peritoneal metastasis nodules. Lower panels: mesenteric metastasis nodules. (E) Quantification of peritoneal metastasis nodules counts and total tumor weight, n = 7 for SKOV3-Vector, n = 6 for SKOV3-OVE-CD38, * p < 0.05, ** p < 0.01). The proportion of mesenteric metastases nodules was evaluated using Fisher's exact test (* p < 0.05). (F) Tumor formation in C57BL/6 mice subcutaneously injected with ID8 cells. After 4 weeks, Compound 78c were administered intraperitoneally (10 mg/kg/dose) every two days for 3 weeks. Tumors were excised and photographed. (G) Quantification of tumor volume in the transplanted tumors ( n = 5 per group, * p < 0.05, ** p < 0.01). (H) Flow cytometry detected proportion of CD38-positive TCs and TILs, CD4/8 + T cells, macrophages, and PD-L1-positive cells in the transplanted tumors ( n = 3 per group, * p < 0.05, ** p < 0.01).

Article Snippet: Four weeks post-inoculation, the CD38-specific inhibitor Compound 78c (S8906, Selleck Chemicals, TX, USA) was administered to C57BL/6 by intraperitoneal injection (10 mg/kg/dose [ ]) every two days over a period of 3 weeks.

Techniques: In Vivo, Injection, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry

Journal: Translational Oncology

Article Title: CD38 contributes to tumor progression and tumor microenvironment reshaping in epithelial ovarian cancer

doi: 10.1016/j.tranon.2025.102414

Figure Lengend Snippet: CD38 promotes ovarian cancer tumorigenesis through PI3K-AKT and IL-6 pathway (A) KEGG enrichment plot of CD38 involvement in JAK-STAT and PI3K-AKT pathways. (B) qPCR analysis of relative core gene mRNA expression in the PI3K-AKT pathway in A2780 cells stably expressing vector or CD38 (Vector or OVE-CD38), normalized to β-actin ( n = 3 per group, *** p < 0.001, **** p < 0.0001). (C) qPCR analysis of relative CD38 and core gene mRNA expression in the PI3K-AKT pathway in subcutaneous tumors from C57BL/6 mice treated with Compound 78c, normalized to β-actin ( n = 3 per group, * p < 0.05, ** p < 0.01, **** p < 0.0001). (D) Western blot analysis of core protein expression levels in the PI3K-AKT pathway in A2780 cells stably expressing vector or CD38 (Vector or OVE-CD38), and CAOV3 cell lines stably expressing shNC or shCD38 (shNC or shCD38). (E) Luminex liquid suspension chip assay for 27 chemokines levels in SKOV3-Vector or SKOV3-OVE-CD38 mouse subcutaneous tumor tissues. (F) qPCR analysis of relative core gene mRNA expression in the IL6 pathway in subcutaneous tumors from C57BL/6 mice treated with Compound 78c, normalized to β-actin ( n = 3 per group, * p < 0.05, ** p < 0.01, **** p < 0.0001). (G) Comparative expression analysis of IL6 pathway core genes between high and low CD38-expressing groups in the GSE9891 dataset (*** p < 0.001, **** p < 0.0001).

Article Snippet: Four weeks post-inoculation, the CD38-specific inhibitor Compound 78c (S8906, Selleck Chemicals, TX, USA) was administered to C57BL/6 by intraperitoneal injection (10 mg/kg/dose [ ]) every two days over a period of 3 weeks.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Luminex, Suspension

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A CD38 expression in CTCL was assessed using data from Nielsen et al. 2021 ( GSE143382 ), comparing relative CD38 expression in skin biopsy samples from CTCL patients ( N = 70) to healthy donors ( N = 12) (log fold change 4.8; p < 0.0001 by Mann–Whitney test). B Single-cell RNA sequencing analysis from previously published datasets ( GSE128531 , GSM5280111 , GSE165623 ) [ – ] compared CD38 cell expression in cells from healthy human skin ( N = 4) to CTCL patient skin ( N = 7). C CD38 expression in CTCL cell lines H9, HH, Hut78, and Hut102 was analyzed by flow cytometry. D Formalin-fixed, paraffin-embedded (FFPE) skin biopsies from CTCL patients ( N = 6) and healthy donor skin ( N = 1) were stained for CD38 and imaged at original magnification x4 and x20 using a Cytation5 imager and Gen5 software.

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: Expressing, MANN-WHITNEY, RNA Sequencing, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Staining, Software

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A Schematic representation of the experimental design for in vivo testing. CD38+ luciferase expressing CTCL cells (H9 line) were engrafted intravenously in immunodeficient NRG mice which were randomly assigned to treatment conditions with either αCD38 antibody (daratumumab; 100 mg/kg subcutaneously weekly for four weeks) or IgG isotype control (0.8 mg/kg subcutaneously once a week for four weeks). Tumor burden was measured over time using IVIS imaging and quantified using Living Image software. B Representative images of mice treated with isotype or αCD38 antibody and quantification of tumor burden total flux (photons/second) at 19 days post-engraftment. αCD38 antibody treatment resulted in an average total flux = 1.4e7 photons/sec ( N = 4), while IgG average total flux = 9.0e7 photons/sec ( N = 3); p = 0.0002. C Flow cytometry analysis of tumor cells isolated from the bone marrow of mice, performed 28 days post-engraftment after three weeks of treatment with either IgG isotype control ( N = 5) or αCD38 ( N = 7). Lymphocytes were gated for human CD45+ cells and analyzed for human CD38 signal. D Quantification of the percentage of CD45+ tumor cells expressing CD38 based on the data from panel ( C ) ( p < 0.0001 by unpaired t test). E qPCR analysis of the relative expression of CD38 , B-catenin ( CTNNB1 ), TCF7 , and BCL6 genes in CTCL tumor cells isolated from the bone marrow of mice that were treated with either isotype or αCD38 antibody ( p < 0.0001 in all conditions by 2way ANOVA).

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: In Vivo, Luciferase, Expressing, Control, Imaging, Software, Flow Cytometry, Isolation

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A Schematic demonstrating development process of CD38 KO H9 CTCL cell line using CRISPR-Cas9, including flow cytometry showing post-sorting purity and CD38- status of CD38 KO line (figure made with Biorender). B CD38 KO CTCL cells showed no growth differences compared to CD38 WT ( p = 0.22 by linear regression). C Migration assay comparing CD38 WT and CD38 KO CTCL cells with and without CXCL12 chemoattractant ( p < 0.01 by unpaired t test). D Western blot of β-catenin, Bcl6, and Tcf7 protein levels with β-actin loading control comparing between H9 CD38 WT and H9 CD38 KO luciferase CTCL cell lines and healthy donor CD4 + T-cells (HD CD4) imaged via chemiluminescence with a FluorChem Imager and images processed with AlphaView software.

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: CRISPR, Flow Cytometry, Migration, Western Blot, Control, Luciferase, Software

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A Comparative assessment of total intracellular NAD+ and NADH levels between CD38 WT and CD38 KO CTCL cells using relative luminescence units (RLU) revealed significantly higher levels in CD38 KO cells ( p < 0.0001 by unpaired t test). B Separate measurements of NAD+ vs NADH levels in CD38 WT and CD38 KO CTCL cells by RLU indicated significantly elevated NAD+ levels in CD38 KO cells ( p < 0.0001 by unpaired t test). C Analysis of the NAD + /NADH ratio displayed a notable increase in CD38 KO cells ( p = 0.0006 by unpaired t test). D Seahorse XF Cell Mito Stress Test assay compared oxygen consumption rate (OCR) between the CD38 WT and CD38 KO CTCL cell lines (normalized to pmol/min/µg protein). E Quantification of the average baseline normalized OCR as well as maximal OCR from the seahorse assay in panel D indicated a significant increase in both baseline and maximal OCR in CD38 KO cells compared to CD38 WT cells (baseline OCR showed a 2.26-fold increase, p < 0.0001; maximal OCR exhibited a 1.83-fold increase, p < 0.0001 as determined by the Mann–Whitney test). F Seahorse XF Cell Mito Stress Test assay compared OCR of CD38 WT cells treated with CD38 inhibitor compound 78c (0.5 µM overnight) or a DMSO control. G Quantification of baseline and maximal normalized OCR between CD38 inhibitor-treated cells and DMSO control cells demonstrated a significant increase in both baseline and maximal OCR in the CD38 inhibitor-treated cells (baseline OCR 1.38-fold increase, p < 0.0001; maximal OCR exhibited a 1.19 fold increase, p = 0.021 as determined by Mann–Whitney test).

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: MANN-WHITNEY, Control

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A Intravenously engrafted H9 CD38 WT and H9 CD38 KO CTCL cell lines in immunodeficient NOD Rag −/− γc −/− (NRG) mice were monitored over time using an In Vivo Imaging System (IVIS; Perkins-Elmer) and representative images from day 18 post-engraftment are shown. B Quantification of the tumor cell luminescent intensity signal as measured by total flux (photons/second) in the CD38 WT and CD38 KO intravenous cohorts (CD38 WT = 2.2e8 photons/sec average total flux, N = 7; CD38 KO = 1e9 photons/sec, N = 5; p = 0.003 by Mann–Whitney test). C Flow cytometry analysis of human CD45+ tumor cells harvested the bone marrow of NRG mice post IV-engraftment and expansion of H9 CD38 WT and H9 CD38 KO luciferase CTCL cell lines. D IVIS images of tumor signal at 19 days post-subcutaneous flank engraftment of H9 CD38 WT and H9 CD38 KO luciferase CTCL cell lines in NRG mice. E Tumor cell burden as measured by total flux of subcutaneous CD38 WT and CD38 KO cells (CD38 WT = 1.01e8 photons/sec average total flux, N = 7; CD38 KO = 6.01e9 photons/sec, N = 8; p < 0.0001 by unpaired t test). F Flank tumors were dissected and photographed on a grossing board. Image J was used to measure tumor size in mm 2 ( p = 0.01 by Mann–Whitney test). G Dissected tumors were formalin fixed and paraffin embedded before being stained for human CD38 and imaged at original magnification x4 and x20 on a Cytation5 imager with Gen5 software.

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: In Vivo Imaging, MANN-WHITNEY, Flow Cytometry, Luciferase, Staining, Software

Journal: Leukemia

Article Title: CD38 in the pathobiology of cutaneous T-cell lymphoma and the potential for combination therapeutic intervention

doi: 10.1038/s41375-025-02551-4

Figure Lengend Snippet: A CTCL cells were treated with one of several histone deacetylase inhibitor agents followed by staining for CD38 and subsequent analysis via flow cytometry using a BD LSRFortessa (figure made with Biorender). B The mean fluorescent intensity of CD38 expression on CTCL cells treated for 72 h with either 1 μM vorinostat, 1 nM romidepsin, 25 nM of panobinostat, or DMSO control (panobinostat vs DMSO p < 0.001 by unpaired t test). C Representative histograms displaying CD38 expression at 72 h for increasing doses of panobinostat (5 nM, 10 nM, and 25 nM) compared to an isotype control. D Median fluorescence intensity (MFI) of CD38 expression on H9 cells treated with increasing doses of panobinostat for across multiple time points (24 h, 48 h, and 72 h) (max 85% increase in CD38 expression with 25 nM panobinostat vs. DMSO at 72 h; p < 0.0001 by 2way ANOVA). E Experimental design and treatment regimen for the in vivo combination αCD38 antibody and panobinostat in mice engrafted H9 luciferase CTCL cells. The animals were divided into four age and sex-matched experimental groups ( N = 4 for all groups): Vehicle and IgG; Vehicle and αCD38 antibody; Panobinostat and IgG; and Panobinostat and αCD38 antibody. Panobinostat (20 mg/kg) and vehicle (2% DMSO, 48% PEG300, 2% Tween80, and 48% ddH2O) were administered via IP injection twice a week, while αCD38 antibody (daratumumab 100 mg/kg) and IgG isotype control (0.8 mg/kg) were administered subcutaneously once a week for three weeks. Mice were monitored and survival was tracked (figure made with Biorender). F Survival curve depicting the outcomes of the four experimental groups in the αCD38 antibody and panobinostat combination study (vehicle/IgG median survival 23 days; vehicle/ αCD38 antibody median survival 32 days; Panobinostat/IgG median survival 27.5 days; combination median survival 39 days; p = 0.01 by log-rank test).

Article Snippet: The CD38 inhibitor 78c compound (Selleckchem, #S8960) was used at a concentration of 0.5uM overnight.

Techniques: Histone Deacetylase Assay, Staining, Flow Cytometry, Expressing, Control, Fluorescence, In Vivo, Luciferase, Injection

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet: CD38 inhibition promotes central memory cell formation and counteracts CAR-T cell exhaustion to enhance antitumor efficacy (A) Schematic depicting in vitro culture model. CAR-T cells were cocultured with NALM6 at the E:T ratio of 1:1 for 48 h, followed by CD38 inhibitor treatment (78C 10 μM, RBN013209 50 μM, luteolinidin 10 μM) for 72 h. Cell differentiation, activation, and exhaustion status were evaluated by flow cytometry. (B) Flow cytometric analysis of CD62L and CD45RO in each group. (C) Frequency of naive cells (CD62L + , CD45RO – ), central memory cells (CD62L + , CD45RO + ), effector memory cells (CD62L – , CD45RO + ), and effector cells (CD62L – , CD45RO – ) in control or inhibitor-treated CAR-T cells 12 days after T cell activation (n = 5 biological replicates). Two-tailed Student’s unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, no significance. Tn, naive T; Tcm, central memory T; Tem, effector memory T; Teff, effector T. Statistical comparison is between each inhibitor-treated group with control. (D) Frequency of CD25 + and CD69 + CAR-T cells in control or inhibitor-treated groups 12 days after T cell activation (n = 5 biological replicates). (E) Frequency of LAG-3 + , TIM-3 + , and PD-1 + CAR-T cells in control or inhibitor-treated groups 12 days after T cell activation (n = 6, 3 biological replicates with two technical replicates for each donor). (F) Frequency of inhibitory receptor co-expression (LAG-3, TIM-3, and PD-1) in control or 78C-treated groups (n = 3 biological replicates). (G) Expansion kinetics of control and 78C-treated CD19-41BBz CAR-T cells during in vitro setting. Arrows indicate the time point of inhibitor treatment and drug washout (n = 3 technical replicates from one donor). (H) Flow cytometric analysis of annexin V in each group. (I) Frequency of apoptosis (annexin V + ) in control or 78C-treated CAR-T cells 12 days after T cell activation (n = 6, 3 biological replicates with 2 technical replicates for each donor). Two-tailed Student’s unpaired t test. (J) Specific lysis of NALM6-luciferase after coculture with control and 78C-treated CAR-T cells upon multiple rounds of tumor challenge. CAR-T cells were cocultured with NALM6-luci cells at the E:T ratio = 1:10 for every 24 h. Data are mean ± standard deviation (SD) of 3 technical replicates from one donor. Specific cytotoxicity is evaluated by (nontransduced T cell viability – CAR-T cell viability)/nontransduced T cell viability × 100%. (K) Specific lysis of NALM6-luciferase after coculture with control and 78C-treated CD19-41BBz CAR-T cells for 72 h at low E:T ratio (n = 3 technical replicates from donor 1 and donor 2, respectively). (L–O) Secretion of granzyme B, IL-2, IFNγ, and TNFα by control and 78C-treated CAR-T cells after the coculture of NALM6-luciferase for 72 h at E:T ratio of 1:1 and 1:128, respectively (n = 3 technical replicates).

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: Inhibition, In Vitro, Cell Differentiation, Activation Assay, Flow Cytometry, Control, Two Tailed Test, Comparison, Expressing, Lysis, Luciferase, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet: Perturbing CD38 boosts the efficacy of CAR-T cells against hematological malignancies in vivo (A) Schematic depicting in vivo experimental setup. NSG mice received 1 × 10 6 NALM6 cells on day −6 and 1.5 × 10 6 either nontransduced T cells (MOCK) or 4-1BB CD19-CAR-T cells on day 0. Bone marrow and spleen tissue were collected for fluorescence-activated cell sorting analysis on day 8. (B) D0-D49 bioluminescence imaging (BLI) imaging of tumor clearance. n = 5 biological replicates for each group. (C) The dorsal BLI signal is displayed for individual mice in each treatment group. n = 7 biological replicates pooled from two independent experiments. (D) BLI imaging of tumor burden on D28 after CAR-T cell infusion. (E) Kaplan-Meier survival plot for mice receiving mock T cells, control CAR-T cells, or CAR-T cells pretreated with CD38 inhibitors. n = 7 biological replicates pooled from two independent experiments (statistical analysis by Mantel-Cox test between control CAR-T and 78C-treated CAR-T group, ∗∗∗p = 0.0007). (F) Absolute numbers of human T cells in the bone marrow (hindlimb) and spleen on day 8 after CAR-T cell injection. n = 3 or more mice per group. (G) Frequency of inhibitory receptor co-expression (LAG-3, TIM-3, and PD-1) in bone marrow CAR-T cells in control (n = 3 biological replicates) or 78C-treated groups (n = 4 biological replicates). (H) Frequency of effector CAR-T cell subset (CD62L – , CD45RO – ) from bone marrow and spleen tissue in control (n = 3 biological replicates) or 78C-treated groups (n = 4 biological replicates). (I) Frequency of CD25 + and CD69 + CAR-T cells from bone marrow and spleen tissue in control (n = 3 biological replicates) or 78C-treated groups (n = 4 biological replicates).

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: In Vivo, Fluorescence, FACS, Imaging, Control, Injection, Expressing

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet: CD38 inhibition results in reduced glycolysis metabolism in CAR-T cells (A) Principal component analysis of CAR-T cells in different groups. (B) Volcano plot illustrating differential gene expression analysis in CD38-inhibited CAR-T compared to control CAR-T cells after coculture with NALM6 cells at 1:1 (E:T). (C) Heatmap of selected pathways enriched in genes significantly upregulated or downregulated in 78C-treated CAR-T cells. A single sample enrichment score was calculated for each pathway, and the mean was taken per response group. A color gradient ranging from blue to red indicates the mean normalized enrichment score (ranging from −2 to +2) of pathways enriched in induced (red) or repressed (blue) genes. (D–F) Metabolic rate as measured by Seahorse analysis of extracellular acidification rate (ECAR) of control (n = 5 technical replicates) or CD38-inhibited (n = 4 technical replicates) CAR-T cells after coculture with NALM6 cells. (G) Heatmap of differentially expressed genes in canonical glycolysis pathway in comparison with the control group. (H) mRNA level of glycolysis-related transcription factors in control or CD38-inhibited CAR-T cells after coculture with NALM6 cells. Data are mean ± SD of 3 technical replicates. (I) Flow cytometric analysis of 2-NBDG uptake in each group. (J) Median fluorescence intensity of 2-NBDG in control or 78C-treated CAR-T cells after coculture with NALM6 cells. Data are mean ± SD of 3 independent experiments from three different donors. (K) Cellular lactate acid level in control or 78C-treated CAR-T cells after coculture with NALM6 cells (n = 4 technical replicates from two different donors). (L) Flow cytometric analysis cytoplasmic ROS in control or 78C-treated CAR-T cells after coculture with NALM6 cells by CM-H2DCFDA staining (n = 3 biological replicates). (M and N) Western blot analysis of PDH (M) and normalized PDH expression relative to TUBULIN in each group (N). NALM6-stimulated CAR-T cells were treated with DMSO/78C for 3 days. Quantitative analysis of western blot data obtained in n = 3 technical replicates is shown, normalized to tubulin.

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: Inhibition, Gene Expression, Control, Comparison, Fluorescence, Staining, Western Blot, Expressing

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet: CD38 inhibition induced CD38-cADPR-Ca 2+ signaling suppression in CAR-T cells (A) Schematic of CD38-cADPR signaling and following activation of Ca 2+ signaling. (B) Intracellular cADPR and ADPR level in control or 78C-treated CAR-T cells after coculture with NALM6 cells (cADPR, n = 10, 5 biological replicates with 2 technical replicates for each donor. ADPR, n = 8, 4 biological replicates with 2 technical replicates for each donor). (C) Median fluorescence intensity of Fluo-4 in CAR-T cells treated with DMSO, 78C, and 78C with cADPR, ADPR, or NAADP after coculture with NALM6 cells (n = 4 biological replicates). (D) Flow cytometric analysis of CD62L and CD45RO in CAR-T cells treated with DMSO, 78C, and 78C with cADPR, ADPR, or NAADP. (E) Frequency of CD62L + CAR-T subset in each group (n = 4 biological replicates). (F) Frequency of CD69 + CAR-T cells in CAR-T cells treated with DMSO, 78C, and 78C with cADPR, ADPR, or NAADP (n = 3 biological replicates). (G) Frequency of LAG-3 + , TIM-3 + , and PD-1 + subsets in CAR-T cells treated with DMSO, 10 μM 78C, and 10 μM 78C with 30 μM cADPR, 500 μM ADPR, or 100 μM NAADP. (H) mRNA level of glycolysis-related transcription factors in CAR-T cells treated with DMSO, 10 μM 78C, or 10 μM 78C with 5 μM cADPR (n = 3 biological replicates). (I) Flow cytometric analysis of CD62L and CD45RO in CAR-T cells treated with DMSO or 10 μM 8-Br-cADPR. (J) Frequency of CD62L + CAR-T subset in each group (n = 3 donors). (K) Frequency of CD25 + and CD69 + CAR-T cells in control or 8-Br-cADPR-treated CAR-T cells after coculture with NALM6 cells (n = 3 biological replicates). (L) Frequency of LAG-3 + , TIM-3 + , and PD-1 + subsets in control or 8-Br-cADPR-treated CAR-T cells after coculture with NALM6 cells (n = 3 biological replicates). (M) mRNA level of glycolysis-related transcription factors in CAR-T cells treated with DMSO or 8-Br-cADPR (n = 3 technical replicates).

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: Inhibition, Activation Assay, Control, Fluorescence

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet: CD38-NAD + -SIRT1 signaling is enhanced upon CD38 inhibition in CAR-T cells (A) Schematic of CD38-NAD-SIRT1-HIF1a signaling. (B and C) Total NAD+ level and the ratio of NAD + to NADH in control or 78C-treated CAR-T cells after coculture with NALM6 cells (n = 4 biological replicates). (D and E) Western blot analysis of SIRT1 (D) and normalized SIRT1 expression relative to ACTIN in each group (E). CAR-T cells were treated with DMSO/78C for 3 days after coculture with NALM6 cells. Quantitative analysis of western blot data obtained in n = 5 experiments from three donors is shown, normalized to β-actin. (F) SIRT1 activity in control or 78C-treated CAR-T cells after coculture with NALM6 cells (n = 3 biological replicates). (G) mRNA level of HIF1A transcription factor in control or 78C-treated CAR-T cells after coculture with NALM6 cells (n = 3 biological replicates). (H and I) Cell lysates from control or 78C-treated CAR-T cells were subjected to immunoprecipitation with a HIF1A antibody followed by western blot analysis with lysine acetylation or HIF1A antibodies. Data are presented as the means ± SD of 6 technical replicates from three different donors. (J) Frequency of naive cells, central memory cells, effector memory cells, and effector cells in CAR-T cells treated with DMSO, 10 μM 78C, or 10 μM 78C plus 100 μM IOX2 (n = 3 biological replicates). (K) Frequency of CD69 + CAR-T cells in each group (n = 3 biological replicates). (L) Frequency of LAG-3 + , TIM-3 + , and PD-1 + CAR-T cells in each group (n = 3 biological replicates). (M) mRNA level of glycolysis-related transcription factors in each group of CAR-T cells (n = 3 technical replicates).

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: Inhibition, Control, Western Blot, Expressing, Activity Assay, Immunoprecipitation

Journal: Cell Reports Medicine

Article Title: Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism

doi: 10.1016/j.xcrm.2024.101400

Figure Lengend Snippet:

Article Snippet: Small molecule drugs including compound 78C (10μM, Selleck, S8960), RBN013209 (50μM, MCE, HY-144987), Luteolinidin Chloride (10μM, Targetmol, TN1895), 8-Bromo-cADP-Ribose (8-Br-cADPR) (8uM, SANTA, sc-201514A), cADP-Ribose (cADPR) (30μM, Sigma, C7344), IOX2 (100μM, Selleck, S2919), Adenosine 5′-diphosphoribose sodium (ADPR sodium) (500μM, MCE, HY-100973A), Nicotinic acid adenine dinucleotide phosphate sodium salt (NAADP sodium) (100μM, sigma, N5655), Dehydronitrosonisoldipine (3uM, MCE, HY-Z0816), DSRM-3716 (30μM, MCE, HY-W021879) and Ned19 (20μM, 50uM, Targetmol, T12205).

Techniques: Recombinant, CCK-8 Assay, Isolation, Bicinchoninic Acid Protein Assay, Extraction, Enzyme-linked Immunosorbent Assay, Activity Assay, Sequencing, Gene Expression, Software