Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism

Abstract

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

Figure 1. Kras^G12D Inactivation Leads to Rapid Tumor Regression

(A) Kaplan-Meier overall survival analysis for mice of indicated genotypes. Cohort size for each genotype is indicated. Arrow: time point for starting doxy treatment. On: mice were fed with doxy-containing water from 3 weeks of age. Off: mice were maintained doxy-free. (B) Histopathological characterization of PDAC from iKras p53^L/+ mice showing (i) typical ductal adenocarcinoma with well-differentiated glandular tumor cells and strong stromal reaction; (ii) local invasion of tumor cells (right of the dotted line) into duodenum wall; (iii) liver metastasis (arrow); and (iv) lung metastasis (arrow). (C) H&E staining shows histological changes of PDAC at the indicated time points after doxy withdrawal. Scale bar represents 100 μm. (D) MRI scan illustrating tumor (area within the dotted line) shrinkage following 7 days of doxy withdrawal. (E) ¹⁸FDG-PET/CT scan of tumor bearing mice at day 0 and day 7 upon doxy withdrawal. *, tumor; Kd, kidney; Bd, bladder dome. See also Figure S1.

Figure 2. Histopathological Characterization of Tumor Regression upon Kras^G12D Inactivation

(A) iKras p53^L/+ mice were fed with doxy-containing water for 9 weeks from 3 weeks of age. The mice were pulled from doxy treatment at the indicated days and injected with BrdU. Pancreatic tumors were stained with antibodies to BrdU (panels i, iv, vii, x), cleaved-caspase3 (panels ii, v, viii, xi), and SMA (panels iii, vi, ix, xii). (B) Pancreatic tumors from (A) were stained with antibodies to phospho-Erk (panels i, iii, v, vii) and phospho-S6 (panels ii, iv, vi, viii). Scale bar represents 100 mm. See also Figure S2.

Figure 3. Transcriptional Changes Induced by Kras^G12D Inactivation

(A) Orthotopic xenograft tumors were generated from five independent primary iKras p53^L/+ PDAC cell lines. Animals were kept on doxy for 2 weeks until tumors were fully established. Half of the animals were pulled off doxy for 24 hr at which point tumor lysate was prepared. Ras activity was measured with Raf-RBD pull-down assay. (B) Heat maps of the genes enriched in indicated metabolism pathways illustrate the changes in gene expression upon doxy withdrawal. Expression levels shown are representative of log₂ values of each replicate from either xenograft tumors or cultured parental cell lines. Red signal denotes higher expression relative to the mean expression level within the group and green signal denotes lower expression relative to the mean expression level within the group. (C) GSEA plot of steroid biosynthesis (top), pyrimidine metabolism (middle), and O-glycan biosynthesis (bottom) pathways based on the off-doxy versus on-doxy gene expression profiles. NES denotes normalized enrichment score. See also Figure S3.

Figure 4. Kras^G12D Extinction Leads to Decreased Glucose Uptake and Glycolysis

(A) Summary of the changes in glycolysis upon Kras^G12D inactivation. Metabolites that decrease upon doxy withdrawal are indicated with green arrows. Bar graphs indicate the relative expression levels of differentially expressed glycolytic enzymes that showed a significant decrease in the absence of doxy; the gene names for those enzymes that exhibited significant changes are also highlighted in the cartoon in blue. Glycolytic enzymes whose change in expression was not significant are illustrated in gray. (B) Heat map of those metabolites that are significantly and consistently changed upon doxy withdrawal between the two iKras p53^L/+ lines as determined by targeted LC-MS/MS using SRM. Cells were maintained in the presence or absence of doxy for 24 hr, at which point metabolite levels were measured from triplicates for each treatment condition. The averaged ratios of off-doxy over on-doxy levels for differentially regulated metabolites are represented in the heat map. Asterisks indicate metabolites involved in glucose metabolism that decrease upon doxy withdrawal. (C and D) iKras p53^L/+ cells were maintained in the presence or absence of doxy for 24 hr. Relative changes of glucose (C) or lactate (D) levels in the medium were measured and normalized to cell numbers. (E) Fold changes of glycolytic intermediates upon doxy withdrawal for 24 hr. *p < 0.05; **p < 0.01. Error bars represent SD of the mean. B(1,3)PG, 1,3-bisphosphoglycerate; B(2,3)PG, 2,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate. See also Figure S4.

Figure 5. Kras^G12D Inactivation Leads to Inhibition of the Hexosamine Biosynthesis Pathway and Protein O-Glycosylation

(A) Summary of changes in the HBP upon Kras^G12D inactivation. Metabolites that decrease upon doxy withdrawal are indicated with green arrows. Differentially expressed genes upon doxy withdrawal are highlighted in blue. (B) Fold change of metabolites in the HBP upon doxy withdrawal for 24 hr. (C and D) Relative mRNA (C) and protein levels (D) of Gfpt1 in the presence or absence of doxy for 24 hr. (E) Western blot analysis for O-linked N-acetylglucosamine (O-GlcNAc) levels in cells maintained in the presence or absence of doxy for 24 hr. For control samples, MEFs were cultured in the presence or absence of glucose for 24 hr. (F) Western blot analysis for O-GlcNAc and Gfpt1 levels in cells infected with shRNA against GFP or Gfpt1. (G and H) Clonogenic assay (G) and soft-agar colony formation assay (H) for iKras p53^L/+ or LSL-Kras p53^L/+ PDAC cells infected with shRNA against GFP or Gfpt1. (I) iKras p53^L/+ cell lines were infected with shRNA against GFP or Gfpt1 and subcutaneously injected into nude mice. Tumor volumes were measured and data shown are representative of results from three independent cell lines. *p < 0.05; **p < 0.01. Error bars represent SD of the mean. GlcNAc-1P, N-acetylglucosamine 1-phosphate; GlcNAc-6P, N-acetylglucosamine 6-phosphate; GlcN6P, glucosamine 6-phosphate; UDP-GlcNAc, UDP-N-acetylglucosamine. See also Figure S5.

Figure 6. Kras^G12D Preferentially Enhances Nonoxidative PPP to Support Ribose Biogenesis

(A) Summary of changes in the PPP upon Kras^G12D inactivation. Metabolites that decrease upon doxy withdrawal are indicated with green arrows. Differentially expressed genes upon doxy withdrawal are highlighted in blue. (B) iKras p53^L/+ cells were maintained in the presence or absence of doxy for 24 hr, at which point U-¹³C glucose labeling kinetics for the indicated metabolites were compared at 1, 3, and 10 min. (C) Fold changes for metabolites in the PPP upon doxy withdrawal for 24 hr. (D) Relative mRNA levels of PPP genes in the presence or absence of doxy for 24 hr. (E) iKras p53^L/+ cells were maintained in the presence or absence of doxy for 24 hr, followed by a 24 hr labeling with 1-¹⁴C or 6-¹⁴C glucose. Incorporation of radioactivity into DNA or RNA were determined and normalized to DNA or RNA concentration. (F) iKras p53^L/+ cells were infected with shRNA against Rpia and Rpe individually or in combination. shRNA against GFP was used as a control. Cells were labeled with 1-¹⁴C glucose and incorporation of radioactivity into DNA and RNA was determined as in (E). (G) iKras p53^L/+ cells were maintained under high (11 mM) or low (1 mM) glucose, and clonogenic activity was determined for cells infected with shRNA against GFP, Rpia, or Rpe. (H) Quantification of colony numbers. (I) iKras p53^L/+ cells were infected with shRNA against GFP, Rpia, or Rpe and subcutaneously injected into nude mice. Tumor volumes were measured and data shown are representative of results from three independent cell lines. Error bars represent SD of the mean. *p < 0.05; **p < 0.01. E4P, erythrose 4-phosphate; GδL6P, 6-phosphoglucono-δ-lactone; GSH, reduced glutathione; GSSG, oxidized glutathione; 6PG, 6-phosphogluconate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; SBP, sedoheptulose 1,7-bisphosphate; X5P, xylulose 5-phosphate. See also Figure S6.

Figure 7. MAPK Pathway Is Critical for Kras^G12D-Mediated Metabolism Reprogramming

(A) iKras p53^L/+ cells were treated with AZD8330 (50 nM), BKM120 (150 nM), or Rapamycin (20 nM) for 18 hr. In parallel, cells were cultured in the presence or absence of doxy for 24 hr to serve as controls and relative mRNA levels of indicated metabolism genes were measured by QPCR. Error bars represent SD of the mean. *p < 0.05; **p < 0.01. (B) Schematic representation of the shift in glucose metabolism upon Kras^G12D withdrawal. Activation of oncogenic Kras enables PDAC tumor maintenance through the increased uptake of glucose and subsequent shunting into glycolysis, the HBP pathway (to enable enhanced glycosylation) and the nonoxidative arm of the PPP (to facilitate ribose biosynthesis for DNA/RNA). Glucose flux into the oxidative arm of the PPP and the TCA cycle do not change when Kras^G12D is inactivated.