Fig 2: The loss of pyruvate carboxylase elicits mitochondrial protein hyperacetylation.A, Western blot of acetylated proteins from 9-week-old fed and 24 h fasted Pcxf/f and PcxL-/- mice. B, Western blot of acetylated proteins from 9-week-old fed, 24 h fasted, 12 week high fat diet–fed, and 1 week ketogenic diet–fed Pcxf/f and PcxL-/- mice. C, quantitative TMT acetyl-proteomics of 24 h fasted Pcxf/f and PcxL-/- livers showing mitochondrial proteins (n = 5). D, quantitative TMT acetyl-proteomics of 24 h fasted Pcxf/f and PcxL-/- livers showing all proteins (n = 5). E, Western blots of mitochondrial proteins from 9-week-old Pcxf/f and PcxL-/- mice (n = 4). Pyruvate Carboxylase (Pcx), Pyruvate Dehydrogenase 1a (Pdh1a), Citrate Synthase (Cs), Malate Dehydrogenase 2 (Mdh2), Mitochondrial Pyruvate Carrier 2 (Mpc2), Glutamine Oxaloacetate Transaminase 2 (Got2), Carnitine Palmitoyltransferase 2 (Cpt2), Acyl-CoA Dehydrogenase Medium Chain (Acadm), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (Hadha), Acyl-CoA Thioesterase 2 (Acot2), ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (Atp5a), ubiquinol cytochrome c reductase core protein 2 (Uqcrc2), mitochondrially encoded cytochrome c oxidase I (Mtco1), succinate dehydrogenase complex, subunit B (Sdhb), NADH:ubiquinone oxidoreductase subunit B8 (Ndufb8).

Fig 3: GOT2 positively regulates PPARd activity. A, IHC staining for GOT2 or GOT2 and panCK in pancreas tissues from KrasLSL-G12D/+;Pdx1-Cre (KC) mice at 6 or 12 months of age (representative of n = 4 per time point). Scale bars = 20 µm. B, IHC staining for GOT2 or GOT2 and panCK in human PDAC (representative of n = 5). Fluorescent images: scale bar = 5 µm, brightfield image: scale bar = 20 µm. Arrowheads indicate examples of tumor cells with nuclear GOT2. C, Scatter plot depicting the correlation of GOT2 expression with expression of PPARd target genes in human PDAC per TCGA RNA-seq data (n = 177). FPKM, fragments per kilobase of transcript per million mapped reads. D, Luciferase assay for PPRE activity in the indicated cell lines, normalized to renilla, presented as mean ± SEM. ****, P < 0.0001 by one-way ANOVA (688M) or an unpaired t test (FC1245). E, PPARd transcriptional activity assay, reading out binding to immobilized DNA containing PPREs, in the indicated cell lines. Data are presented as mean ± SEM from four (FC1245) or three (8988T) independent experiments. **, P < 0.01 by an unpaired t test. F and G, ChIP for H3K9Ac (F) and PPARd (G) in control or sgGot2 688M PDAC cells, followed by qPCR for proximal promoter regions of the indicated genes. Data were normalized to an intergenic region (int. B) and are presented as mean ± SEM from biological triplicates. ns = not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by an unpaired t test. H, qPCR for the indicated PPARd-regulated genes in control or GOT2-knockdown PDAC cells, treated with vehicle (DMSO) or the PPARd synthetic agonist GW501516 (GW; 100 nmol/L). Data are presented as mean ± SEM from biological triplicates. ns = not significant. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by one-way ANOVA. I, Luciferase assay for PPRE activity in sgGOT2 PDAC cells reconstituted with wild-type GOT2 (wtGOT2) or NLS-wtGOT2. Data are presented as mean ± SEM from four independent experiments. ns = not significant. ****, P < 0.0001 by one-way ANOVA. J, PDAC tumor weight at the experimental endpoint, 22 days after orthotopic transplantation of FC1245 cells into immune-competent hosts (n = 4–5 per arm). Data are presented as mean ± SEM. ns = not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA. K–M, IHC staining and quantification of T-cell markers CD3, CD8, and CD4 in FC1245 tumors (n = 4–5 per arm, scale bars = 20 µm). Data are presented as mean ± SEM. ns = not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA. N, IHC staining and quantification of CD68 and Arg1 in FC1245 tumors (n = 4–5 per arm, scale bars = 20 µm). Data are presented as mean ± SEM. ns = not significant. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA.

Fig 4: PPARd activation restores tumor growth and T-cell exclusion in the absence of GOT2. A, Viable cell measurements in control or sgGot2 PDAC cells treated with vehicle or 100 nmol/L GW501516. RLU, relative light unit. B, PDAC tumor weight at the experimental endpoint, 30 days after orthotopic transplantation of the control or sgGot2 cells, with daily i.p. injection of vehicle or 4 mg/kg GW501516. Ctrl: n = 5 per cohort, sgGot2: n = 4 per cohort. Data are presented as mean ± SEM. ns = not significant. *, P < 0.05; **, P < 0.01 by one-way ANOVA. C, IHC staining of control and sgGot2 688M tumors treated with vehicle or GW501516 as in B for the T-cell marker CD3. Representative images are shown above (scale bars = 50 µm), with quantification below (ctrl: n = 5, ctrl + GW501516: n = 5, sgGot2: n = 3, sgGot2 + GW501516: n = 4). Data are presented as mean ± SEM. ns = not significant. **, P < 0.01 by one-way ANOVA. D, IHC staining for PTGS2/COX2 in control or sgGot2 PDAC treated with vehicle or GW501516 (representative of n = 3–5 per cohort). Scale bars = 50 µm. E, Viable cell measurements in control or sgGot2 PDAC cells stably transduced with empty vector or VP16–PPARd. Data are presented as mean ± SEM. F and G, PDAC tumor weight at the experimental endpoint in the indicated 688M (F) and FC1245 (G) lines. 688M: Ctrl: n = 5, sgGot2 a: n = 4, ctrl VP16–PPARd: n = 4, sgGot2 a VP16–PPARd: n = 4, endpoint = day 27. FC1245: Ctrl: n = 5, sgGot2 a: n = 5, ctrl VP16–PPARd: n = 5, sgGot2 a VP16–PPARd: n = 4, endpoint = day 18. Ctrl and sgGot2 FC1245 arms here are also depicted in Fig. 1E. ns = not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA. H, qPCR for PPARd-regulated genes in the indicated FC1245 stable cell lines, normalized to 36b4. Data are presented as mean ± SEM from biological triplicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by one-way ANOVA. I, PDAC tumor weight at experimental endpoint (day 18) in ctrl (n = 4) and shPpard (n = 5 per hairpin) FC1245 tumors. **, P < 0.01 by one-way ANOVA. J, Quantification of CD3 IHC on the tumors from I (scale bars = 50 µm). ns = not significant. *, P < 0.05 by one-way ANOVA. K, Heat map depicting differentially expressed (DE) genes in control and sgGot2 FC1245 PDAC cells, untreated or treated with 500 nmol/L GW501516 for 24 hours (n = 3 per group), identified by RNA-seq using cutoff criteria Padj < 0.01 and logFC < -1 or > 1 in at least one comparison. L, Venn diagram showing RNA-seq results by the criteria in K, with sample gene identities at the overlap listed. Overlapping gene frequency: ***, P < 0.001 by permutation test. M, Molecular Signatures Database (MSigDB) pathway analysis showing the top 10 enriched pathways of genes at the overlap in L.

Fig 5: GOT2 promotes pancreatic tumor progression without impacting proliferation. A, Viable cell measurements in the indicated PDAC cell lines. Data are presented as mean ± SEM from biological triplicates. ****, P < 0.0001 by one-way ANOVA. Dox, doxycycline; RLU, relative light unit. Numbers in parentheses are the short hairpins (shRNAs) used for the GOT2 knockdowns. B, PDAC tumor weight at the experimental endpoint, 34 days after orthotopic transplantation of 688M cells into immune-competent hosts. Ctrl: n = 8, sgGot2 a: n = 9, sgGot2 b: n = 10. Data are presented as mean ± SEM. ns = not significant. ****, P < 0.0001 by one-way ANOVA. C, IHC staining of tumors in B for Ki-67 (proliferation) and pan-cytokeratin (panCK; tumor cells), with a DAPI counterstain (nuclei). Representative images are shown on the left (scale bars = 50 µm), with quantification on the right (ctrl: n = 6, sgGot2: n = 5). Data are presented as mean ± SEM. ns = not significant by an unpaired t test. D, PDAC tumor weight at the experimental endpoint, 22 days after orthotopic transplantation of FC1245 cells into immune-competent hosts. Ctrl: n = 5, shGot2 a: n = 5, shGot2 b: n = 3. Data are presented as mean ± SEM. ns = not significant. **, P < 0.01; ***, P < 0.001 by one-way ANOVA. E, PDAC tumor weight at the experimental endpoint, 18 days after orthotopic transplantation of FC1245 cells into immune-competent hosts. Ctrl: n = 5, sgGot2 a: n = 5. Data are presented as mean ± SEM. ****, P < 0.0001 by an unpaired t test.