Targeting DGAT1 reprograms lipid landscape and restores CD8⁺ T cell immunity in pancreatic cancer

Figures

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  Fig. 1. CRISPR screening identifies DGAT1 as a metabolic checkpoint in PDAC. A Schematic of the integrated computational workflow used to analyze human PDAC scRNA-seq datasets, linking tumor-intrinsic metabolic pathway activity with CD8⁺ T cell functional and differentiation states. Top enriched metabolic pathways in tumor cells are shown on the right. B Parallel in vitro and in vivo CRISPR screens using a metabolism-focused sgRNA library. MAGeCK analysis and robust rank aggregation (RRA) identified top genes selectively depleted in tumors grown in immunocompetent C57BL/6 mice compared with in vitro cultures. Two-sided tests were used, and P values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) method. C, D Dgat1 knockdown in subcutaneous KPCmut tumors in nude and C57BL/6 mice. Tumor volume (C) and weight (D) are shown. Data represent mean ± SD; n = 8 mice per group (nude) and n = 6 mice per group (C57BL/6). Two-way ANOVA (tumor growth) and one-way ANOVA (tumor weight). E Kaplan–Meier survival analysis of C57BL/6 mice orthotopically implanted KPCmut tumors. Pancreas weight at the endpoint is shown on the right. Data represent mean ± SD; n =…

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  Fig. 2. DGAT1 expression is elevated in PDAC and correlates with poor prognosis. A–C Representative multiplex immunofluorescence staining and quantification of DGAT1 expression in tissues from human pancreatic tissues (cohort #1) (n = 60). DGAT1 expression was stratified by tumor stage (B) or pathological characteristics (C). Scale bars, 0.5 mm. H-score, histological score. Data represent mean ± SD; two-tailed t-test. D Relative DGAT1 mRNA expression in different cell subtypes in human normal pancreatic tissues and PDAC tumors (GSA: CRA001160). E DGAT1 mRNA expression in normal pancreatic tissues (n = 362) and PDAC tumors (n = 178) in TCGA datasets. Two-tailed t-test. F Violin plot showing the correlation of DGAT1 mRNA expression with PDAC subtypes: basal-like (n = 39) and classical (n = 130). Normalized RNA-seq expression data were obtained from EGAS00001002543. Two-tailed t-test. G Western blot analyses of DGAT1 protein expression in KrasG12D-ON or -OFF AK14837 cell line. KRAS expression was suppressed upon doxycycline withdrawal. H Representative multiplex immunofluorescence staining and quantification of DGAT1 and CD8 expression in tissues from human pancreatic tissues (cohort #1)…

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  Fig. 3. DGAT1 inhibition reprograms the TME and promotes stem-like CD8⁺ T cell differentiation. A Schematic illustration of xenograft tumorigenesis assays inoculated with KPCflox cells and application of antibodies for CD8+ and CD4+ T cell depletion. B, C Effect of Dgat1 knockdown on subcutaneously implanted KPCflox tumor growth in C57BL/6 mice treated with isotype IgG, anti-CD4 or anti-CD8 antibodies. Tumor volume and weight were shown. Data represent mean ± SD; n = 6 mice per group. Two-way ANOVA for tumor growth and one-way ANOVA for tumor weight. D scRNA-seq was performed on CD8+ T cells isolated from KPCflox tumors. UMAP plot depicting the distribution of clusters, colored by annotated cell type in both groups. Relative fraction of each cluster was shown on the right. Tumors were pooled from 5 mice per group. E Pseudotime trajectory analysis of CD8+ T cells using scVelo, with cells color-coded by CD8+ T cell subtypes and pseudotime progression. F Visualization of gene signatures scores of memory, progenitor and exhaustion gene sets in CD8+ T cells. G Gene set enrichment analysis for indicated pathways in intratumoral CD8+ T cells isolated from KPCflox tumors. H, I Flow cytometric…

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  Fig. 4. DGAT1 loss rewires lipid metabolism and promotes lipid uptake in tumor cells. A, B Effect of murine H426A mutant on subcutaneously implanted KPCflox tumor growth in C57BL/6 mice. Tumor volume (A) and weight (B) were shown. Data represent mean ± SD; n = 6 biologically independent samples. Two-way ANOVA for tumor growth and one-way ANOVA for tumor weight. C Heatmap of representative lipids in AsPC-1 cells analyzed by LC-MS/MS. FFA, free fatty acids; HexCer, hexose ceramides; BMP, Bis(monoacylglycero)phosphate; Cer, ceramide; SM, sphingomyelins; CL, cardiolipins; PE, phosphatidylethanolamines; PS, phosphatidylserines; PG, phosphatidylglycerols; PI, phosphatidylinositols; PC, phosphatidylcholines; CE, cholesteryl esters; DAG, diacylglycerols; TAG, triacylglycerols. D Representative images of lipid droplets in indicated cancer cells stained with BODIPY 493/503, analyzed by fluorescent imaging. Cells were pre-treated with or without DGAT1 inhibitor A-922500 (30 µM) for 48 h. Scale bar 10 µm. E Schematic illustration of isotope tracing experiments. AsPC-1 cells were cultured in DMEM supplemented with 5% lipid-free fetal bovine serum and 0.2 mM [U-13C16]palmitic acid for 6 h. F Relative…

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  Fig. 5. DGAT1 loss restricts FFA overload and boosts CD8+ T cell fitness and stemness. A GSEA analysis shows the altered pathways for the loss of DGAT1 in tumor-infiltrating CD8+ T cells. NES, normalized enrichment score. B, C Representative fluorescence images, quantification lipid droplets and fluorescent intensity in CD8+ T cells stained with BODIPY 493/503. CD8+ T cells were isolated from Dgat1 knockdown or control KPCflox tumors 3 weeks after inoculation. Data represent mean ± SD; n = 4 biologically independent samples per group; two-tailed t-test. D, E Heatmap and quantification of representative total or individual fatty acids in interstitial fluid derived from KPCflox-tumors, quantified by LC-MS/MS. Data represent mean ± SD; n = 4 biologically independent samples per group; two-tailed t-test. F Schematic illustration of coculture assay with indicated fatty acid supplements. G Flow cytometric analysis of expression of memory (TCF1+, CD62L and IL-7Rα)-associated markers and the ratio of Tpex in CD8+ T cells treated with indicated fatty acid supplements (200 µM). Data represent mean ± SD; n = 3 biologically independent samples per group; one-way ANOVA. H Schematic illustration of…

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  Fig. 6. DGAT1 inhibition preserves stem-like CD8⁺ T cell states via FOXO1 activation. A Chromatin accessibility heatmap grouped by differential accessibility patterns of CD8+ T cells, isolated from KPCflox tumors. Tumors were pooled from 5 mice per group. B Normalized gene expression of FOXO1 regulon genes projected onto the UMAP plot. Violin plots depicts normalized expression of Sell and FOXO1 regulon genes across cell clusters. C Chromatin accessibility tracks for the Tcf7 and Ccr7 gene loci of intratumoral CD8+ T cells, isolated from KPCflox tumors. FOXO1 chromatin immunoprecipitation followed by sequencing (ChIP–seq) peaks are aligned with the corresponding gene loci. D GSEA analysis shows the enrichment of FOXO1 regulon genes in DGAT1-deficient CD8+ TILs isolated from KPCflox tumors. E Flow cytometric analysis of FOXO1 expression in CD8+ T cells treated with indicated fatty acid supplements (200 µM). Data represent mean ± SD; n = 3 biologically independent samples per group; one-way ANOVA. F Immunofluorescence staining of FOXO1 in CD8+ T cells treated with BSA or palmitic acid (200 µM). n = 50 cells per group. Cyto, Nuc and Nuc/Cyto denote FOXO1 expression in the cytoplasm, nucleus…

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  Fig. 7. DGAT1 inhibition potentiates the efficacy of immune checkpoint blockade. Survival curve of orthotopically implanted KPCmut (A) or KPCflox (B) tumor-bearing mice treated with anti-PD-1 or IgG control antibodies (200 µg per mouse, every 3 days), followed by detecting macroscopic and microscopic tumors after autopsy (C). Pancreas weight at the endpoint is shown on the right. Data represent mean ± SD; n = 7 mice per group, except the combination treatment group (n = 9). Log-rank (Mantel–Cox) test (survival) and one-way ANOVA (pancreas weight). Survival curve of orthotopically implanted KPCmut (D) or KPCflox (E) tumor-bearing mice treated with DGAT1 inhibitor A-922500 (80 mg/kg/day, oral gavage) and/or anti-PD-1 antibodies. Pancreas weight at the endpoint is shown on the right. Data represent mean ± SD; n = 6 mice per group, except in KPCflox (E) mice: A-922500 + IgG (n = 7) and A-922500 + α-PD-1 (n = 9). Log-rank (Mantel–Cox) test (survival) and one-way ANOVA (pancreas weight). F Flow cytometric analysis of expression of memory (TCF1+, CD62L and IL-7Rα)-associated markers in CD8+ TILs isolated from KPCflox tumors. Data represent mean ± SD; n = 6 biologically independent samples per…